DNA vaccines against foot-and-mouth disease virus

ABSTRACT

This application is directed generally to foot-and-mouth disease virus (FMDV) 3C proteases that have been modified by mutating a polynucleotide sequence coding for the FMDV 3C protease. The modified FMDV proteases exhibit proteolytic activity on FMDV P1 precursor protein and exhibit a reduction in one or more toxic or inhibitory properties associated with an unmodified FMDV 3C protease on a host cell used to recombinantly produce it. Vectors carrying polynucleotides encoding modified FMDV 3C protease sequences can induce production of FMDV virus-like particles in a host cell when expressed in the host cell. The modified FMDV 3C proteases can generally be used to produce immunogenic FMDV preparations capable of inducing an immune response against FMDV.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.15/259,409, filed Sep. 8, 2016, the contents of which is hereinincorporated by reference in its entirety.

GOVERNMENT RIGHTS

This invention was made with government support under HSHQPM-12-X-00013and HSHQDC-14-F-00035 awarded by the U.S. Department of HomelandSecurity. The United States Government has certain rights in thisinvention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. The ASCII copy is namedDHS-078US03_Sequence_Listing.txt and is 611 KB in size.

BACKGROUND Field of the Invention

The present disclosure relates to foot-and-mouth disease virus (FMDV) 3Cproteases that have been modified by mutating a polynucleotide sequencecoding for the FMDV 3C protease. The modified FMDV proteases exhibitreduced cytotoxicity when expressed in host cells.

Description of Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentdisclosure.

The foot-and-mouth disease virus (FMDV), a prototypic aphthovirus withinthe Picornaviridae family, is the causative agent of a highly infectiousand sometimes fatal disease that affects cloven-hoofed animals such ascattle, pigs, sheep, goats, deer and other animals with divided hooves.There are seven major FMDV antigenically distinct virus serotypes (A, O,C, Asia 1 and South African Territories or SAT 1, 2 and 3) and multiplesubtypes or topotypes exist within each serotype. Infection with any oneserotype does not confer protective immunity against another. Serotype 0is the most common serotype worldwide.

After an animal is infected with FMDV, the first signs of illnessusually appear within 2 to 14 days. These usually include high fever for2-3 days followed by blisters inside the mouth and on the feet that mayrupture and cause lameness.

FMD outbreaks cause significant agro-economic losses and have severeimplications for animal farming throughout much of the world. Forexample, the estimated costs attributable to the 2001 outbreak of FMD inthe U.K. were £ 8 billion, including the costs slaughtering andsanitarily disposing of 6 million livestock. The virus causing thedisease is highly contagious and can be spread by direct contact orthrough aerosols to uninfected livestock by infected livestock or bydomestic or wild animals. FMDV may be also transmitted by contact withcontaminated farming equipment, vehicles, clothing, or feed.Consequently, the containment of FMDV demands considerable effort andexpense required for vaccination, vigilance and strict monitoring oflivestock, or culling and disposal of infected livestock, as well as foraccommodating transport and trade restrictions, quarantines and otheradministrative and legal issues.

The current most commonly used FMD vaccines utilize whole virus that hasbeen killed, inactivated, and/or attenuated. A whole virus vaccineincludes dozens of FMDV antigens to provide a broad spectrum of immunityagainst different FMDV strains and variants, including those arising dueto antigenic drift or antigenic shift. However, this vaccine platform isfraught with many limitations and shortcomings. Animals immunized withthe whole virus are difficult to distinguish from infected animals alsoexposed to the whole virus. The efficacy of the current vaccineformulations is limited by immunogenic instability and short vaccineshelf life that results in a loss of potency upon transporation orstorage and subsequent induction of insufficient immunity or immunity ofa short duration. Furthermore, the set-up and running costs of producingthe current FMDV vaccine in potent form and securing and maintaining itsproduction facilities are very high. For example, the mode of producingcurrent FMDV vaccine raises safety concerns due to the possibility ofvirus escape from a vaccine production facility. Recombinant productionof FMDV antigens, which could avoid the problems inherent to use ofwhole virus-based vaccines, is impeded by the promiscuous proteolyticactivity of the FMDV 3C protease which is required for the processing ofFMDV antigens from the FMDV P1 precursor polypeptide, but which alsocleaves proteins in host cells used to express recombinant FMDVantigens. Native FMDV 3C protease is toxic to host cells andsignificantly reduces their ability to express immunogenic FMDV antigensin significant amounts.

There is a continuing need and interest in the development of new secondor third generation vaccines that generate strong and stable protectiveimmune responses against FMDV. With these objectives in mind, theinventors developed modifications to the FMDV 3C protease so that it cancleave and process FMDV precursor polypeptides to produce immunogenicFMDV antigens, but does not exert significant cytotoxic effects on thehost cells used to express FMDV immunogens.

BRIEF SUMMARY OF THE INVENTION

The inventors have surprisingly discovered that the 3C protease can beengineered to maintain its ability to process and cleave the foot andmouth disease P1 polypeptide precursor into separate virus proteins andto simultaneously reduce its toxic, inhibitory, and other deleteriouseffects on a host cell used to express FMDV proteins which result inpoor recombinant yields of P1 and processed viral proteins. Even moresurprisingly, this has been accomplished by modifying a surface regionof the FMDV 3C protease that is distal from the 3C active site andsubstrate binding cleft.

One aspect of the invention involves engineering a polynucleotideencoding a modified foot-and-mouth disease (FMDV) 3C protease whichcomprises one or more amino acid substitutions within residues 26-35,125-134 or 138-150 of a wild-type FMDV 3C protease. The engineeredpolynucleotide may encode a modified FMDV 3C protease having additionalsubstitutions outside of the residues described above, may encode atruncated 3C protease with terminal or internal deletions that do notremove its proteolytic activity, or may encode additional proteins orpeptide or polypeptide segments besides the modified 3C protease, suchas FMDV P1 precursor protein, one or more FMDV viral proteins, such asVP0, VP1, VP2, VP3, VP4, 2A, Δ1D2A, translation interrupter sequences,markers, reporters, or tags such as luciferase of FLAG or poly-His, ortranscriptional or translational regulator elements. In one non-limitingembodiment, the engineered polynucleotide is produced by introducing oneor more nucleotide point mutations into the codons encoding residues26-35, 125-134 or 138-150 of a wild-type FMDV 3C protease and/or intoother residues forming a wild-type B₂ β-strand of the 3C protease, forexample, by mutating the codon encoding the leucine residue at position127 to encode a proline residue (L127P).

An associated aspect of the invention is a vector or polynucleotideconstruct that comprises or contains a polynucleotide encoding anengineered polynucleotide containing one or more modified FMDV 3Cproteases. Such a vector or polynucleotide construct may also compriseone or more polynucleotide sequences encoding FMDV P1 precursor protein,one or more FMDV viral proteins, such as VP0, VP1, VP2, VP3, VP4, 2A,Δ1D2A, translation interrupter sequences, markers or tags such asluciferase or FLAG. The vector may also comprise an origin ofreplication, one or more selectable markers, such as antibioticresistance genes, as well as at least one promoter or othertranscription regulatory element, prokaryotic or eukaryotic translationinitiation sequence or other translation regulatory element,translational interrupter sequence, or reporter gene operatively linkedto the polynucleotide sequence encoding the modified FMDV 3C protease orthe FMDV P1 precursor polypeptide.

Another associated aspect of the invention is a host cell that has beentransformed or transfected with a polynucleotide encoding the modifiedFMDV 3C protease or with the vector or polynucleotide constructsdescribed above and that is capable of expressing the FMDV 3C protease,and in some embodiments, both the FMDV 3C protease and a FMDV P1precursor polypeptide. In some embodiments the host cell will expressboth the modified FMDV 3C protease and the FMDV P1 precursorpolypeptide, and permit processing of the FMDV P1 precursor polypeptideinto viral proteins, such as FMDV VP0, VP3, and VP1 or into VP1, VP2,VP3 and VP4. A host cell according to an aspect of the invention mayalso be selected or engineered to provide for transport, secretion orassembly of recombinantly expressed viral proteins into quaternarystructures such as virus-like particles.

Another aspect of the invention involves the recombinant production ofFMDV P1 precursor polypeptide and processed viral proteins derivedtherefrom by action of the modified FMDV 3C protease. Such a methodgenerally involves culturing a host cell according to an aspect of theinvention in a suitable medium and recovering FMDV P1 precursorpolypeptide, processed products of P1 such as VP0, VP1, VP2, VP3 or VP4,or processed and assembled quaternary structures of FMDV viral proteins,such as FMDV virus-like particles. Such methods are superior toconventional recombinant methods for expression of FMDV P1 or FMDV viralproteins because the modified FMDV 3C protease expressed is less toxicor inhibitory to host cell growth and expression of FMDV recombinantproducts. Such a method may also involve expression of a marker orindicator such as a luciferase that can be secreted to monitor orquantify expression of FMDV 3C protease, P1 precursor polypeptide orFMDV viral proteins or a tag to facilitate purification of suchproducts.

The methods employing polynucleotide constructs or vectors encoding 2Aor 2A-like protein sequences and/or Gaussia Luciferase (GLuc) orSecreted Gaussia luciferase (SGLuc) sequences represent another aspectof the invention. Such constructions may be employed to express andprocess polyproteins translated from an open reading frame such as butnot limited to the FMDV P1 precursor protein. Individual proteinsprocessed from longer proteins, such as precursor proteins, may beseparately targeted to and quantified in different cellular orextracellular compartments. For example, SGLuc may be translated as partof a precursor protein adjacent to 2A or a 2A-like sequence whichseparates SGLuc from the polyprotein allowing it to be secreted from thehost cell. The amount of luciferase secreted into the extracellularmedium by a host cell may be used to detect, monitor or quantify theamount of transgene expression in the host cell. Polynucleotidesexpressing proteins comprising GLuc or SGLuc with, or without,translation interrupter sequences may be used to express proteins havingluciferase activity. Such fusion proteins may comprise a GLuc 8990mutation that stabilizes SGLuc luciferase expression in cell lysisbuffer (30).

A modified FMDV 3C protease is another aspect of this invention. Such amodified protease will exhibit proteolytic activity on FMDV P1 precursorprotein and will generally exhibit a reduction in one or more toxic orinhibitory properties associated with an unmodified FMDV 3C protease ona host cell used to recombinantly produce it. As described above, themodified FMDV 3C protease according to an aspect of the inventioncomprises one or more amino acid substitutions within residues 26-35,125-134 or 138-150 of a wild-type FMDV 3C protease, but may containadditional modifications outside of these segments and may exhibit atleast one proteolytic activity on FMDV P1 precursor polypeptide. In oneor more embodiments, the modified FMDV 3C protease may contain cysteineresidues at positions 51 and 163, contain FMDV 3C protease residues H46,D84 and C163, or the following substitutions or features: 126E, R126E orany non-native residue at position 126, A133, A133S or any non-nativesubstitution at position 133. Other substitutions at residues 26-35, 46,80, 84, 125-134, 138-150, 163 or 181 of a native 3C protease amino acidsequences may be made. These include, but are not limited to C163A,C163G, C163S, R126P, I128L, I128P, H46Y, H181Y, D80E, D84E, or D84N orcombinations to two, three of more of these substitutions with otherspecific substitutions described herein. In other embodiments, thenative bridging cysteine residues or all native cysteine and prolineresidues of the 3C protease are retained.

Proteolytic, antigenic or immunogenic products or compositionscomprising the recombinant FMDV 3C protease or FMDV viral proteins orquaternary structures, such as FMDV virus-like particles, representanother aspect of the invention. Such compositions may contain one ormore buffers suitable for activity of the modified FMDV 3C protease, orcontain carriers, adjuvants, immune enhancers or other excipientssuitable for administration of FMDV proteins, quaternary structures ofFMDV viral proteins, or virus-like particles to a subject in needthereof. Such subjects generally include mammals susceptible to FMDVinfection and may also include other animal or biological vectors ofFMDV infection.

A cellular immunogen or vaccine comprising a host cell, preferably, anautologous, syngeneic or allogeneic host cell, that has been transformedto express the FMDV 3C protease according to the invention and,preferably, one or more other FMDV antigens, such as FMDV P1 precursorpolypeptide represents another aspect of the invention. In oneembodiment, the host cell may be administered to a subject as a livecell, attenuated cell (e.g., irradiated, fixed or chemically-treated),or a cell that has been disrupted or fractionated (e.g., lysed,sonicated, French-pressed, sheared, freeze-thawed, or emulsified). Inone embodiment, the immunogen or vaccine may be a host cell that whenadministered to a subject expresses in vivo a modified 3C protease andat least one FMDV antigen, such as P1 precursor polypeptide. Other kindsof host cells expressing modified 3C protease and/or P1 precursorpolypeptide or other FMDV immunogens may also be administered, such aslive yeast or bacterial cells capable of persisting in a subject'sgastrointestinal tract.

Another aspect of the invention is a method for inducing an immuneresponse against FMDV in a subject in need thereof, who may or may nothave been exposed to FMDV. Such a method may include administration ofat least one FMDV P1 precursor polypeptide, P1 polypeptide, VP0, VP1,VP2, VP3 or VP4 protein, a quaternary structure of FMDV virus proteins,or FMDV virus-like particles, such as those produced by the action ofthe modified FMDV 3C protease on an FMDV P1 precursor polypeptide. Thismethod also includes administration of a cellular immunogen or cellularvaccine as described above. It may also involve administering a vectoror polynucleotide construct encoding a FMDV P1 precursor polypeptide andmodified 3C protease, which when expressed in the muscle or othersomatic cells of an immunized subject preferably assemble intoimmunogenic quaternary or VLP structures. Such a method may also includeadministering a vector or polynucleotide construct encoding at least oneof a FMDV P1 precursor polypeptide, P1 polypeptide, VP0, VP1, VP2, VP3or VP4 protein, components of a FMDV virus-like particle or any otherFMDV proteins or immunogens. A nucleic acid based vaccine may be linear,circular, supercoiled, and/or single or double stranded DNA or RNA. Inone embodiment, a vector or polynucleotide construct encoding animmunogen or the vaccine according to the invention may be administeredto a subject in need thereof along with a suitable carrier or adjuvant,for example, into muscle tissue.

The foregoing paragraphs provide a general introduction and are notintended to limit the scope of the following embodiments and claimswhich are best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

An appreciation of the disclosure and many of the attendant advantagesthereof may be understood by reference to the accompanying drawings.Included in the drawings are the following figures:

FIG. 1. The top line depicts the single open-reading frame (ORF) andassociated genetic elements of a picornavirus RNA genome which encodesthe viral polyprotein in a single ORF shown below it. The lower part ofFIG. 1 describes processing of the polyprotein into intermediateprecursor polypeptides and ultimately into individual viral proteins.

FIG. 2 is a ribbon diagram of an FMDV 3C protease crystal structuredescribed by Protein Database identification 2j92, FMDV 3C^(pro) strainA10₆₁ which is incorporated by reference. The ribbon diagram depicts twon-barrel domains which are identified on each side of the diagram, aβ-ribbon identified at the top center of the diagram and the location ofthe substrate binding cleft also identified at the top center of thediagram. The native FMDV 3C protease contains a catalytic triad ofresidues: H46, D84 and C163. FIG. 2 depicts the positions of these threecatalytic residues. Position 163, a cysteine residue in native3Cprotease is labelled as A163 instead of C163. This substitution wasmade to remove 3C proteolytic activity to facilitate isolation ofsufficient intact 3C protease for crystallographic analysis.

FIG. 3 identifies the positions of the triad of catalytic residues atpositions 46, 84 and 163 in the amino acid sequence of the 3C proteaseof wild-type FMDV strain Asia Lebanon 89 (SEQ ID NO: 1). The relativepositions of these catalytic residues are aligned with the knownsecondary structures of the 3C protein which are identified above theamino acid sequence.

FIGS. 4A and 4B illustrate how the Δ1D2A translational interruptersequence allows for expression of two separate peptides, GLuc and FMDV3C protease, from a single open reading frame upon translation. FIG. 4Adescribes the amino acid sequence of the Δ1D2A translation interruptersequence (SEQ ID NO: 120) which is involved in production of separateGLuc (a luciferase) and the 3C protease polypeptides. FIG. 4B provides aschematic diagram of this process and shows the relative position of theΔ1D2A sequence. During translation, the Δ1D2A sequence causestranslational interruption which results in expression of a GLucportion, which is secreted from a host cell into a cell medium, and the3C protease portion both derived from the same ORF. Translationinterruption induced by Δ1D2A occurs prior to the terminal proline (P)residue of the Δ1D2A sequence as shown by the arrow in FIG. 4A. Upontranslation of this chimeric GLuc-2A protein, 29 amino acids of thetranslated Δ1D2A sequence remain attached to the C-terminus of GLuc anda single proline (P) residue from 2A remains attached to 3C as shown bythe lighter box adjacent to the 3C protease on the lower schematicdiagram in FIG. 4B.

FIG. 5 shows the positions of residues substituted by the inventors toproduce modified FMDV 3C proteases. These include V28K, L127P, V141T,C142T and C163A. Residue C163 is one residue of the catalytic triad andits substitution removes 3C protease activity. An example of such aC163A mutant is described by SEQ ID NOS: 209-210. These positions areshown on a ribbon diagram of the FMDV 3C protease crystal structuredescribed by Protein Database identification 2j92, FMDV 3C^(pro) strainA10₆₁ which is incorporated by reference.

FIG. 6 compares luciferase activity of HEK293-T cells that have not beentransfected (first bar), or have been transfected with vectorsexpressing GLuc-3C constructs (bars 2-7). The constructs depicted bybars 2-7 respectively contain the following substitutions to the 3Cprotease sequence of Asia Lebanon 89: V141T (bar 2), wild-type (bar 3,no substitution), V28K (bar 4), C142T (bar 5), L127P (bar 6), and C163A(bar 7) where the FMDV 3C was derived from FMDV serovar Asia Lebanon 89.Features of GLuc-3C constructs are depicted in FIG. 4B. Luciferaseactivity levels are represented in terms of relative light units (RLU)per unit of time (RLU/0.5 s.) Relatively high levels of luciferaseactivity were observed for constructs expressing 3C protease modified atC142T, L127P and C163A.

FIG. 7 diagrams a construct comprising the P1 precursor protein, the 3Cprotease, Δ1D2A translation interrupter, and SGLuc. The lower part ofFIG. 7 shows the relative positions of individual viral proteins VP0,VP1, VP2, VP3, VP4 and 2A within the FMDV P1 polypeptide precursor. Thesite of translation interruption between 3C and SGLuc is indicated bythe light gray Δ1D2A arrow on the top diagram and the sites ofproteolytic P1 precursor protein cleavage by FMDV 3C protease are markedby dark arrows at the bottom of FIG. 7.

FIG. 8 compares luciferase activity of HEK293-T cells that have not beentransfected (first bar), or have been transfected with vectorsexpressing P1-3C-SGLuc constructs (bars 2-7) where the FMDV P1 wasderived from FMDV serovar 01 Manisa and the FMDV 3C was derived fromFMDV serovar Asia Lebanon 89. The constructs depicted by bars 2-7respectively contain the following substitutions to the 3C proteasesequence: V141T (bar 2), wild-type (bar 3, no substitution), V28K (bar4), C142T (bar 5), C163A (bar 6) and L127P (bar 7). Luciferase activitylevels are represented in terms of relative light units (RLU) per unitof time (RLU/0.5 s.) Similar to the constructs in FIG. 6, high levels ofluciferase activity were observed for constructs expressing 3C proteasemodified at C142T, C163A and L127P.

FIGS. 9A-9G compare Western Blots of HEK-293-T cells expressing a2A-SGLuc construct not expressing P1-3C (FIG. 9A), and P1-3C-SGLucconstructs expressing wild-type Asia Lebanon 1989 3C protease (FIG. 9B),and 3C modified at V28K (FIG. 9C), L127P (FIG. 9D), V141T (FIG. 9E),C142T (FIG. 9F) and at C163A (FIG. 9G). As indicated above the lanes ofeach figure, Western blots were probed with antibodies to VP2, VP3 orVP1. The P1 component was derived from the 01 Manisa serovar of FMDVwhile the 3C component was derived from the Asia Lebanon 1989 serovar ofFMDV.

FIG. 9A is a Western blot image of lysate of HEK-293-T cells transfectedwith a 2A-SGLuc construct as a negative control for the examination ofthe ability of an FMDV 3C protease to process and cleave the P1polypeptide protein precursor.

FIG. 9B is a Western blot image of lysate of HEK-293-T cells transfectedwith a O1P1-3C (wild-type)-SGLuc construct for the examination of theability of the wild-type FMDV 3C protease to process and cleave the P1polypeptide protein precursor.

FIG. 9C is a Western blot image of lysate of HEK-293-T cells transfectedwith a O1P1-3C(V28K)-SGLuc construct for the examination of the abilityof the V28K mutant FMDV 3C protease to process and cleave the P1polypeptide protein precursor.

FIG. 9D is a Western blot image of lysate of HEK-293-T cells transfectedwith an O1P1-3C(L127P)-SGLuc construct for the examination of theability of the L127P mutant FMDV 3C protease to process and cleave theP1 polypeptide protein precursor.

FIG. 9E is a Western blot image of lysate of HEK-293-T cells transfectedwith a O1P1-3C(V141T)-SGLuc construct for the examination of the abilityof the V141T mutant FMDV 3C protease to process and cleave the P1polypeptide protein precursor.

FIG. 9F is a Western blot image of lysate of HEK-293-T cells transfectedwith a O1P1-3C(C142T)-SGLuc construct for the examination of the abilityof the C142T mutant FMDV 3C protease to process and cleave the P1polypeptide protein precursor.

FIG. 9G is a Western blot image of lysate of HEK-293-T cells transfectedwith an O1P1-3C(C163A)-SGLuc construct, as a negative control for theexamination of the ability of an FMDV 3C protease to process and cleavethe P1 polypeptide protein precursor.

FIG. 10A is a transmission electron microscope (TEM) image at 3000×magnification of HEK293-T cells expressing the O1P1-3C(wt)-SGLucconstruct. FIG. 10B is a TEM image at 25,000× magnification of HEK293-Tcells expressing the O1P1-3C(wt)-SGLuc construct. Crystal arrays ofvirus-like particles (VLPs) are seen in the center of FIG. 10B. VLPswere conventionally identified by recognition of their crystallinestructure as confirmed by measuring a capsid size characteristic of FMDVVLPs.

FIG. 11A is a TEM image at 10,000× magnification of HEK293-T cellsexpressing the O1P1-3C(C142T)-SGLuc construct. FIG. 11B is a TEM imageat 25,000× magnification of HEK293-T cells expressing theO1P1-3C(C142T)-SGLuc construct. Crystal arrays of virus-like particles(VLPs) are seen in the center of FIG. 11B.

FIG. 12 compares luciferase activity of HEK293-T cells that expresshistone H3 instead of luciferase (first bar), or that have beentransfected with vectors expressing P1-3C-SGLuc constructs (bars 2-7)where the P1 component was derived from the 01 Manisa serovar of FMDVwhile 3C component was derived from the Asia Lebanon 1989 serovar ofFMDV. The constructs depicted by bars 2-6 respectively contain thefollowing substitutions to the 3C protease sequence: wild-type (bar 2,no substitution in 3C), L127P (bar 3), C142T (bar 4), C163A (bar 5) andL127P/C142T (double mutant, bar 6). Luciferase activity levels arerepresented in terms of relative light units (RLU) per unit of time(RLU/0.5 s.) Similar to the luciferase activity of the constructs inFIGS. 6 and 8, higher levels of luciferase activity were observed forconstructs expressing 3C protease modified at L127P, C142T or C163A.Significantly higher levels of luciferase activity were observed for thedouble mutant L127P/C142T.

FIG. 13 is a Western blot image of a lysate of HEK-293-T cellstransfected with an O1P1-3C(L127P/C142T)-SGLuc construct probed withantibodies to VP2, VP3 or VP1. These results show the ability of theL127P/C142T double mutant of FMDV 3C protease to process and cleave theP1 polypeptide protein precursor.

FIG. 14A is a TEM image at 20,000× magnification of HEK293-T cellsexpressing the O1P1-3C(L127P/C142T)-SGLuc construct, showing formationof VLP crystal arrays.

FIG. 14B is a TEM image at 7000× magnification of HEK293-T cellsexpressing the O1P1-3C(L127P/C142T)-SGLuc construct, showing formationof VLP crystal arrays.

FIG. 15 shows that E. coli expressing L127P-(upper row right) orC163A-(upper row left) modified 3C exhibited more bacterial growth thanthose expressing wild-type FMDV 3C (upper row center), C142T-(lower rowleft), or L127P/C142T-(lower row right) modified FMDV 3C protease, asdetermined by bacterial colony count.

FIG. 16 shows that E. coli transformed with constructs expressingL127P-modified 3C protease and C163A-modified 3C protease exhibitedsignificantly higher growth rates as determined by absorbance or opticaldensity (OD₆₀₀) than E. coli transformed with constructs expressingnative, unmodified 3C, C142T-modified 3C or L127P/C142T-modified 3Cprotease. These results suggest that the L127P 3C protease mutant is anexcellent choice for expression of FMDV viral proteins in E. coli.

FIG. 17 shows that the modified L127P-3C protease as expressed in E.coli can proteolytically process FMDV P1 protein co-expressed in E.coli. FIG. 17 is an image of a western blot analysis utilizing anantibody which detects VP2 and showing the ability of the mutant L127PFMDV 3C protease to process the P1 polypeptide precursor in E. coli.Lane 3 depicts His-P1 protein, Lane 6 depicts induced His-P1 proteinwithout induced 3C(L127P), and lane 7 shows processing of induced His-P1by induced 3C(L127P). This His-tag is encoded by the polynucleotidesequence ATGGGCAGCAGCCATCATCATCATCATCACGGC (SEQ ID NO: 204) and has theamino acid sequence MGSSHHHHHHG (SEQ ID NO: 205).

FIG. 18 is a schematic diagram describing the gene layouts of the eightdifferent GLuc/SGLuc-Δ1D2A chimeras prepared and evaluated.

FIG. 19 is a bar graph showing RLU/0.5 second(s) (RLU per 0.5 seconds)luciferase activity levels for equal volumes of harvested supernatantmedium from HEK293-T cells transfected with the eight differentGLuc/SGLuc-Δ1D2A chimeras described by FIG. 18.

FIG. 20A is an image of a Western Blot probed with antibodies to GLucshowing approximately equal protein loading. FIG. 20B is a bar graphshowing RLU/0.5 second luciferase activity levels for theGLuc/SGLuc-Δ1D2A chimeras of FIG. 18 and the histone H3 negativecontrol. FIG. 20B describes the luciferase readings from samples whichhave been adjusted to have approximately equal protein levels asdepicted by western blots shown in FIG. 20A.

FIG. 21 is bar graph showing the percentage of total luciferase activityfor GLuc and SGLuc retained in cell lysis buffers. Luciferase activityin the presence of lysis buffers is significantly higher for SGLuc thanfor GLuc. “LCLB”: Luciferase Cell Lysis Buffer; “MPER”: mammalianprotein extraction reagent.

FIG. 22 is a schematic diagram illustrating of the constructs used totest for the formation of VLPs in the presence of SGLuc.

FIG. 23 is a bar graph showing RLU/0.5 second luciferase activity levelsin extracellular media harvested from LF-BK αV/β6 cells transfected withSGLuc, P1-3C-Δ1D2A-SGLuc, or P1-3C as described in FIG. 22. P1 componentwas derived from the 01 Manisa serovar of FMDV while 3C component wasderived from the Asia Lebanon 1989 serovar of FMDV.

FIGS. 24A-24I depict the results of immunofluorescence (IFA) using the6HC4 antibody, which was used as a negative control since it is specificto FMDV serotypes other than O when used in IFA, and antibodies 12FE9and F21 which are, respectively, specific to FMDV type O VP1 and VP2peptides for all FMDV serotypes. FIG. 24A is an IFA image of a DNAvector expressing SGLuc examined for VLP formation using 6HC4antibodies. FIG. 24B is an IFA image of a DNA vector expressing the P1polypeptide precursor and FMDV 3C protease examined for VLP formationusing 6HC4 antibodies. FIG. 24C is an IFA image of a DNA vectorexpressing the SGLuc, P1 polypeptide precursor and FMDV 3C proteaseexamined for VLP formation using 6HC4 antibodies. FIG. 24D is an IFAimage of a DNA vector expressing SGLuc examined for VLP formation using12FE9 antibodies. FIG. 24E is an IFA image of a DNA vector expressingthe P1 polypeptide precursor and FMDV 3C protease examined for VLPformation using 12FE9 antibodies. FIG. 24F is an IFA image of a DNAvector expressing the SGLuc, P1 polypeptide precursor and FMDV 3Cprotease examined for VLP formation using 12FE9 antibodies. FIG. 24G isan IFA image of a DNA vector expressing SGLuc examined for VLP formationusing F21 antibodies. FIG. 24H is an IFA image of a DNA vectorexpressing the P1 polypeptide precursor and FMDV 3C protease examinedfor VLP formation using F21 antibodies. FIG. 24I is an IFA image of aDNA vector expressing the SGLuc, P1 polypeptide precursor and FMDV 3Cprotease examined for VLP formation using F21 antibodies.

FIGS. 25A-25D illustrate by immunoelectron microscopy (“IEM”) theformation of crystalline arrays indicative of VLP formation intransfected LF-BK αV/β6 cells in FIGS. 25A-25B and FIGS. 25C-25D,respectively. IEM utilizes gold labeled antibodies which localize totarget antigen resulting in dark electron dense areas when examined.FIG. 25A is an IEM image at 5,000× magnification of cells transformedwith a DNA vector expressing the SGLuc, FMDV P1 polypeptide precursorand wildtype FMDV 3C protease examined for VLP formation. FIG. 25B is anIEM image at 25,000× magnification, of the region indicated with a boxin FIG. 25A. FIG. 25C is an IEM image at 5,000× magnification, of a DNAvector expressing the FMDV P1 polypeptide precursor and wildtype FMDV 3Cprotease examined for VLP formation. FIG. 25D is an IEM image at 25,000×magnification, of the region indicated with a box in FIG. 25C.

FIG. 26 is an image showing Western blots of cell-free translations ofthe constructs identified along the X-axis probed with antibody toeIF4AI which is a host cell translation/initiation factor. These datashow that eIF4AI processing was significantly lower for the threeconstructs (lanes 4, 7 and 8) containing the L127P mutation (lane 4),the C163A construct expressing inactive 3C (lane 7), and the constructexpressing the L127P/C142T double mutation (lane 8) when compared towild-type 3C and other 3C mutants.

FIG. 27 is a vector map of mpTarget SAT2P1-3C-SGLuc constructs. Thisvector contains a nucleotide sequence encoding for the P1 polypeptideprecursor from the FMDV SAT2 Egypt 2010 strain, a 2A translationinterrupter sequence, a segment encoding a wild-type or mutant FMDV 3Cprotease (e.g., wild-type, and L127P, C142T, C163A, L127P/C142T FMDV 3Cmutants), and a 2A-SGLuc luciferase reporter gene.

FIG. 28 is a bar graph showing RLU/0.5 second luciferase activity levelsfor untransfected HEK293-T cells (negative control, first bar) andHEK293-T cells transfected with the vectors described by FIG. 27containing either sequences encoding wild-type 3C or 3C mutants L127P,C142T, C163A, or L127P/C142T. Significantly higher levels of luciferaseactivity were observed for cells transfected with vectors encodinginactive 3C (C163A, bar 5) or which carried an L127P mutation (bars 3and 6) compared to cells transfected with vectors encoding wild-type 3Cor C142T.

FIGS. 29A-29C show Western blots of HEK293-T cell lysates expressing thevarious constructs described by FIG. 27 probed with antibodies to VP1(monoclonal 6HC4), VP2 (monoclonal IC888) or VP3 (polyclonal Anti-VP3).Lanes from lysates containing mutant L127P 3C proteins showed thepresence of VP1, VP2 and VP3. No cleavage was observed for controlmutant C163A. P1 cleavage products are illustrated in FIG. 7. FIG. 29Ais an image showing Western blotting of HEK293-T cell lysatestransfected with the various embodiments of the vector of FIG. 27 withmonoclonal anti-VP1 antibody 6CH4. FIG. 29B is an image showing Westernblotting of HEK293-T cell lysates transfected with the variousembodiments of the vector of FIG. 27 with monoclonal anti-VP2 antibodyIC888. FIG. 29C is an image showing Western blotting of HEK293-T celllysates transfected with the various embodiments of the vector of FIG.27 with anti-VP3 antibodies.

FIGS. 30A-30F provide transmission electron microscope (TEM) images ofVLP crystal arrays in HEK293-T-cells transfected with the constructsdescribed by FIGS. 27-29. FIG. 30A is a TEM image of HEK293-T cellsexpressing the SAT2P1-3C(L127P)-SGLuc construct at 20,000×magnification, showing formation of VLP crystal arrays. FIG. 30B is aTEM image of HEK293-T cells expressing the SAT2P1-3C(L127P)-SGLucconstruct at 50,000× magnification, showing formation of VLP crystalarrays. FIG. 30C is a TEM image of HEK293-T cells expressing theSAT2P1-3C(C142T)-SGLuc construct at 20,000× magnification, showingformation of VLP crystal arrays. FIG. 30D is a TEM image of HEK293-Tcells expressing the SAT2P1-3C(C142T)-SGLuc construct at 50,000×magnification, showing formation of VLP crystal arrays. FIG. 30E is aTEM image of HEK293-T cells expressing the SAT2P1-3C(L127P/C142T)-SGLucconstruct at 20,000× magnification, showing formation of VLP crystalarrays. FIG. 30F is a TEM image of HEK293-T cells expressing theSAT2P1-3C(L127P/C142T)-SGLuc construct at 50,000× magnification, showingformation of VLP crystal arrays.

FIG. 31 depicts Western blots utilizing the F14 (anti-VP0/2) and 12FE9(anti-VP1) antibodies to evaluate for processing in bacteria transformedwith different plasmid constructs.

FIG. 32 shows Western blots of samples from bacteria transformed withdifferent plasmid constructs utilized in co-immunoprecipitations withB473M and 12FE9 antibodies. Antibody B473M is specific to Type 0 anddependent upon the presence of secondary structures for reactivity whileantibody 12FE9 is specific to VP1 and will recognize the epitope in bothlinear and folded confirmations. Western blots utilized F14(anti-VP0/2), anti-VP3, and 12FE9 (anti-VP1) antibodies.

FIGS. 33A and 33B depict EM images of bacteria transformed with twoplasmids simultaneously and producing VLP arrays. FIG. 33A depicts EMimages of bacteria at 20,000× magnification, and FIG. 33B depicts EMimages of bacteria at 50,000× magnification. One plasmid encodes for theFMDV P1 polypeptide derived from the FMDV serovar 01 Manisa and thesecond plasmid encodes for the FMDV 3C(L127P) mutant derived from FMDVserovar Asia Lebanon 89.

FIG. 34 depicts expression and processing of FMDV O1 Manisa P1polypeptide by either of two 3C protease mutants, L127P (lane 3) orL127P/C142T (lane 4), that were cloned into a baculovirus expressionvector and expressed in SF21 cells. Specific antibodies recognizedprocessed FMDV P1 components: VP0/VP2 (F14 antibody), VP3 (anti-rabbitVP3), or VP1 (12FE9 antibody).

FIGS. 35A-35D compare Western blots of HEK-293-T cells expressing one offive P1-3C(L127P)-SGLuc constructs wherein each respective P1 componentwas derived from one of FMDV serotypes 01 Manisa (SEQ ID NO: 136), A24Cruzeiro (SEQ ID NO: 138), Asial Shamir (SEQ ID NO: 144), C3 Indrial(SEQ ID NO: 141), and SAT2 Egypt (SEQ ID NO: 140), in order to examinethe ability of the L127P 3C mutant protease (SEQ ID NO: 39) to processand cleave each of the P1 polypeptide protein precursor derived from oneof the aforementioned FMDV serotypes.

FIGS. 36A and 36B provide transmission electron microscope (TEM) imagesof VLP crystal arrays in HEK293-T-cells expressing either theAsiaP1-3C(L127P)-SGLuc (FIG. 36A) or O1P1-3C(L127P)-SGLuc (FIG. 36B)constructs at 50,000× magnification.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Foot and Mouth Disease Virus

A “foot-and-mouth-disease virus” or the acronym FMDV refers to, but isnot limited to, any of the seven major FMDV antigenically distinct virusserotypes, for example serotypes A, O, C, Asia 1 and South AfricanTerritories (SAT) 1, 2 and 3 as well as the multiple subtypes ortopotypes which exist within each serotype.

FMDV Hosts/Animals Susceptible to FMDV Infection

The term “host” refers to a mammalian subject, especially but notlimited to cloven-hooved livestock and wildlife (e.g. cattle, pigs,sheep, goats, water buffalos, yaks, reindeer, deer, elk, llamas,alpacas, bison, moose, camels, chamois, giraffes, hogs, warthogs, kudus,antelopes, gazelles, wildebeests) that are in need of treatment forfoot-and-mouth disease (FMD). Hosts are in need of treatment for FMDwhen they are infected with one or more strains of the FMDV, have beendiagnosed with FMD, or are otherwise at risk of contracting FMDVinfection. Hosts that are “predisposed to” to FMD can be defined ashosts that do not exhibit overt symptoms of FMD but that aregenetically, physiologically, or otherwise at risk of developing FMD.

Mutants

The terms “wild-type” or its acronym “wt”, and native refers to abiological molecule that has not been genetically modified, for example,a nucleotide sequence encoding for an FMDV 3C protease that exists innature and has not been genetically modified, an FMDV 3C proteasetranslated from a coding nucleotide sequence that exists in nature andhas not been genetically modified, a transgene expression cassettecontaining a nucleotide sequence encoding for an FMDV 3C protease thatexists in nature and has not been genetically modified, and a vectorcarrying a mutant nucleotide sequence encoding for an FMDV 3C proteasethat exists in nature and has not been genetically modified or atransgene expression cassette containing a mutant nucleotide sequenceencoding for an FMDV 3C protease that exists in nature and has not beengenetically modified.

The term “mutation” as used herein indicates any genetic modification ofa nucleic acid and/or polypeptide which results in an altered nucleicacid or polypeptide. Mutations include, but are not limited to pointmutations, deletions, or insertions of single or multiple residues in apolynucleotide, which includes alterations arising within aprotein-encoding region of a gene as well as alterations in regionsoutside of a protein-encoding sequence, such as, but not limited to,regulatory or promoter sequences. A genetic alteration may be a mutationof any type. For instance, the mutation may constitute a point mutation,a frame-shift mutation, a nonsense mutation, an insertion, or a deletionof part or all of a gene. In addition, in some embodiments of themodified microorganism, a portion of the microorganism genome has beenreplaced with a heterologous polynucleotide. In some embodiments, themutations are naturally-occurring. In other embodiments, the mutationsare identified and/or enriched through artificial selection pressure. Itmust be noted that all the mutations, modifications or alterationsdescribed in the present disclosure are the result of geneticengineering, and not naturally occurring mutations.

The terms “mutated”, “mutant”, “modified”, “altered”, “variant”, and“engineered” are used interchangeably in the present invention asadjectives describing a nucleotide sequence, a nucleic acid, a proteinor a protease. As a non-limiting example, a “mutated nucleotide sequenceencoding for an FMDV 3C protease” or a “mutant nucleotide sequenceencoding for an FMDV 3C protease” refers to a nucleotide sequenceencoding for an FMDV 3C protease that is modified as defined above to bedifferent from the wild-type nucleotide sequence encoding for an FMDV 3Cprotease which may or may not result in at least one of the following:one or more amino acid substitutions and shift in the open reading framefor the translated peptide product, which may be an FMDV 3C proteasethat folds properly (which may be functional or non-functional) or anon-functional peptide. In another non-limiting example, a “mutated FMDV3C protease”, a “mutant FMDV 3C protease”, a “modified FMDV 3C protease”or an “altered FMDV 3C protease” refers to an FMDV 3C protease expressedfrom a mutated nucleotide sequence encoding for an FMDV 3C proteasewhere the amino acid sequence has been altered, as compared to thewild-type FMDV 3C protease, by one or more amino acid substitutions ordeletion of part of the protease (usually from the C-terminus), whichmay also lead to a change in one or more of the protein/proteaseproperties, including but not limited to protein expression levels(e.g., transgene expression), substrate specificity, proteolyticactivity towards FMDV polypeptide precursors, proteolytic activitytowards host proteins, thermal stability, solubility, etc.

The term “mutant”, when used herein as a noun, depending on the context,or the term “variant” refers to one of the following: a mutantnucleotide sequence encoding for an FMDV 3C protease, a mutant FMDV 3Cprotease, a transgene expression cassette containing a mutant nucleotidesequence encoding for an FMDV 3C protease, and a vector carrying amutant nucleotide sequence encoding for an FMDV 3C protease or atransgene expression cassette containing a mutant nucleotide sequenceencoding for an FMDV 3C protease. As a non-limiting example, a “C163Amutant”, or a “3C(163A) mutant”, depending on the context, refers to oneof the following: a nucleotide sequence encoding for an FMDV 3C proteasehaving the cysteine residue at position 163 being substituted with analanine, an FMDV 3C protease having the cysteine residue at position 163being substituted with an alanine, a transgene expression cassettecontaining a nucleotide sequence encoding for an FMDV 3C protease havingthe cysteine residue at position 163 being substituted with an alanine,and a vector carrying a nucleotide sequence encoding for an FMDV 3Cprotease having the cysteine residue at position 163 being substitutedwith an alanine or a transgene expression cassette containing anucleotide sequence encoding for an FMDV 3C protease having the cysteineresidue at position 163 being substituted with an alanine.

Polynucleotides/Vectors/Constructs/Genetic Expression

A “nucleotide” refers to an organic molecule that serves as a monomer,or a subunit of nucleic acids like DNA and RNA. Nucleotides are buildingblocks of nucleic acids and are composed of a nitrogenous base (e.g., A(adenine), G (guanine), C (cytosine), T/U (thymine/uracil), afive-carbon sugar (ribose or deoxyribose), and at least one phosphategroup. Thus, a nucleoside plus a phosphate group yields a nucleotide.Nucleotides in a nucleotide sequence are commonly indicated based ontheir nitrogenous bases.

A “nucleotide sequence” or a “nucleic acid sequence” is a succession ofletters that indicate the order of nucleotides or nucleic acids within aDNA (using GACT) or RNA molecule (using GACU). A DNA or RNA molecule orpolynucleotide may be single or double stranded and may be genomic,recombinant, synthetic, a transcript, a PCR- or amplification product,an mRNA or cDNA. It may optionally comprise modified bases or a modifiedbackbone. It may comprise a sequence in either a sense or antisenseorientation and it inherently describes its complement.

A “recombinant polynucleotide” is a polynucleotide that is not in itsnative state, e.g., the polynucleotide comprises a nucleotide sequencenot found in nature, or the polynucleotide is in a context other thanthat in which it is naturally found, e.g., separated from nucleotidesequences with which it typically is in proximity in nature, or adjacent(or contiguous with) nucleotide sequences with which it typically is notin proximity. For example, the sequence at issue can be cloned into avector, or otherwise recombined with one or more additional nucleicacid.

An “isolated polynucleotide” is a polynucleotide whether naturallyoccurring or recombinant, that is present outside the cell in which itis typically found in nature, whether purified or not. Optionally, anisolated polynucleotide is subject to one or more enrichment orpurification procedures, e.g., cell lysis, extraction, centrifugation,precipitation, or the like.

A “coding region” or simply, a “region” of a gene consists of thenucleotide residues of the coding strand of the gene and the nucleotidesof the non-coding strand of the gene which are homologous with orcomplementary to, respectively, the coding region of an mRNA moleculewhich is produced by transcription of the gene. A “coding region” of anmRNA molecule also consists of the nucleotide residues of the mRNAmolecule which are matched with an anti-codon region of a tRNA moleculeduring translation of the mRNA molecule or which encode a stop codon.The coding region may thus include nucleotide residues corresponding toamino acid residues which are not present in the mature protein encodedby the mRNA molecule (e.g., amino acid residues in a protein exportsignal sequence).

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Vectors/DNA Constructs/Genetic Expression

A “transgene expression cassette”, a “transgene expression construct”,an “expression cassette”, an “expression construct”, a “construct”, a“chimera”, a “chimeric DNA”, a “DNA chimera” or a “chimeric gene” is anucleic acid sequence that has been artificially constructed to compriseone or more functional units (e.g. promoter, control element, consensussequence, translational frameshift sequence, protein encoding gene etc.)not found together in nature, and is capable of directing the expressionof any RNA transcript in an organism that the cassette has beentransferred to, including gene encoding sequence(s) of interest as wellas non-translated RNAs, such as shRNAs, microRNAs, siRNAs, anti-senseRNAs. A transgene expression cassette may be single- or double-strandedand circular or linear. A transgene expression cassette can beconstructed, inserted or cloned into a vector, which serves as a vehiclefor transferring, replicating and/or expressing nucleic acid sequencesin target cells.

A “promoter” is a region of DNA that initiates transcription of aparticular gene or an expression cassette and is located near thetranscription start sites of genes or expression cassettes, on the samestrand and upstream on the DNA (towards the 5′ region of the sensestrand). A promoter can be about 100 to 1000 base pairs long.

A “vector” is any means by which a nucleic acid can be propagated and/ortransferred between organisms, cells, or cellular components. Vectorsinclude viruses, bacteriophage, pro-viruses, plasmids, phagemids,transposons, cosmids, viral vectors, expression vectors, gene transfervectors, minicircle vectors, and artificial chromosomes such as YACs(yeast artificial chromosomes), BACs (bacterial artificial chromosomes),and PLACs (plant artificial chromosomes), and the like, that are“episomes,” that is, that replicate autonomously or can integrate into achromosome of a host cell. A vector typically contains at least anorigin of replication, a cloning site and a selectable marker (e.g.,antibiotic resistance). Natural versions of the foregoing non-limitingexamples may be isolated, purified, and/or modified so the resultantnatural version is differentiable from the material in its naturalstate. A vector can also be a naked RNA polynucleotide, a naked DNApolynucleotide, a polynucleotide composed of both DNA and RNA within thesame strand, a polylysine-conjugated DNA or RNA, a peptide-conjugatedDNA or RNA, a liposome-conjugated DNA, or the like, that are notepisomal in nature, or it can be an organism which comprises one or moreof the above polynucleotide constructs such as an Agrobacterium or abacterium.

The term “recombinant vector” as used herein is defined as vectorproduced by joining pieces of nucleic acids from different sources.

A “minicircle DNA vector” may be referred to as “minicircle vector” or“minicircle” is a small (usually in the range of 3-4 kb, approximately3-4 kb or usually no larger than 10 kb) circular, episomal plasmidderivative wherein all prokaryotic vector parts (e.g., bacterial originof replication, genes associated with bacterial propagation of plasmids)have been removed. Since minicircle vectors contain no prokaryotic DNAsequences, they are less likely to be perceived as foreign and destroyedwhen they are employed as vehicles for transferring transgenes intotarget mammalian cells.

Transformation

“Transformation” refers to the process by which a vector orpolynucleotide construct is introduced into a host cell. Transformation(or transduction, or transfection), can be achieved by any one of anumber of means including chemical transformation (e.g. lithium acetatetransformation), electroporation, microinjection, biolistics (orparticle bombardment-mediated delivery), or Agrobacterium mediatedtransformation.

“Transfection” refers to the process by which a nucleic acid such as agene cloned inside a vector (DNA or RNA) is delivered into a eukaryotichost cell.

Host Cell

The term “host cell” refers to a prokaryotic (e.g. bacterial) or aeukaryotic cell (e.g. mammalian, insect, yeast etc.) that is naturallyinfected or artificially transfected or transformed with a virus or avector, for example, by vaccination. The virus introduced to the hostcell may be live, inactivated, attenuated or modified, while the vectorintroduced carries a transgene expression cassette that, when expressedin the host cell, may produce viral structural proteins thatself-assemble to form virus-like particles (VLPs). In some cases, a hostcell may be inside of a host or subject and said host or subject may betreated by the administration of nucleic-acid-based vaccine encoding amodified FMDV 3C protease and at least one other FMDV antigen. A hostcell may contain a polynucleotide encoding a modified 3C protease in itsgenomic or episomal DNA. For example, a modified FMDV 3C polynucleotidemay be incorporated into a host cell genome via recombination, by use ofa transposon, or by other recombinant DNA methods well known in the art.FMDV 3C protease and other FMDV antigens may also be expressed from thesame or different plasmids, episomes, or other DNA or RNA constructsinside of a host cell.

A host cell for expression of FMDV 3C, FMDV P1 precursor protein, otherFMDV proteins or antigenic sequences, as well as other proteins ofinterest may be a prokaryotic or eukaryotic cells. The term host cellincludes yeast or fungal host cells, such as those of Saccharomycescerevisiae, or Pichia pastoris; plant host cells, such as those ofArabidopsis thaliana, Chlamydomonas reinhardtii, Glycine max, Nicotianabenthamiana, Nicotiana tabacum, Oryza sativa, or Zea mays; insect cellsor insect cell lines such as those of Spodoptera frugiperda, Drosophilamelanogaster, Sf9, or Sf21; the cells of vertebrates or mammals ormammalian cell lines, such as HEK-293T (human kidney embryo) cell, LF-BK(porcine cell), LF-BK αV/β6, or cells of animals susceptible to FMDVinfection; prokaryotic host cells such as those of gram-positivebacteria including cells of Bacillus, Lactococcus, Streptomyces,Rhodococcus, Corynebacterium, Mycobacterium or gram-negative bacteriasuch as Escherichia or Pseudomonas.

Amino Acids/Proteins/Polypeptide Structures

A “residue” or an “amino acid residue” refers to a specific amino acidwithin the polymeric chain of a peptide, a polypeptide or a protein. Aresidue may be one of the twenty two conventional proteinogenic aminoacid residues (which include selenocysteine and pyrrolysine), a modifiedproteinogenic amino acid residue, or a non-proteinogenic amino acidresidue.

An “amino acid sequence”, a “peptide sequence” or a “protein sequence”refers to the order in which amino acid residues, connected by peptidebonds, like in the chain in peptides and proteins. The sequence isgenerally reported from the N-terminal end containing free amino groupto the C-terminal end containing free carboxyl group. Peptide sequenceis often called protein sequence if it represents the primary structureof a protein. Throughout the present disclosure, an amino acid residuemay be represented by a three-letter code or a single-letter code,including but not limited to Ala (A) for alanine, Arg (R) for arginine,Asn (N) for asparagine, Asp (D) for aspartic acid, Cys (C) for cysteine,Gln (Q) for glutamine, Glu (E) for glutamic acid, Gly (G) for glycine,His (H) for histidine, Ile (I) for isoleucine, Leu (L) for leucine, Lys(K) for lysine, Met (M) for methionine, Phe (F) for phenylalanine, Pro(P) for proline, Ser (S) for serine, Thr (T) for threonine, Trp (W) fortryptophan, Tyr (Y) for tyrosine, Val (V) for valine, Pyl (O) forpyrrolysine, Sec (U) for selenocysteine.

As used herein, a “non-coded amino acid”, a “non-proteinogenic aminoacid”, a “synthetic amino acid” or an “unnatural amino acid” refers toan amino acid that is not naturally encoded or found in the genetic code(DNA or mRNA) of any organism, and has to therefore be synthesized invitro.

A “genetically coded amino acid”, a “coded amino acid” or a “naturalamino acid” refers to an amino acid that is naturally encoded by orfound in the genetic code (DNA or mRNA) of an organism, such as alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,pyrrolysine and selenocysteine.

The term “protein,” “peptide,” or “polypeptide” as used herein indicatesan organic polymer composed of two or more amino acidic monomers and/oranalogs thereof. As used herein, the term “amino acid” or “amino acidicmonomer” refers to any natural and/or synthetic amino acids includingglycine and both D- or L-optical isomers. The term “amino acid analog”refers to an amino acid in which one or more individual atoms have beenreplaced, either with a different atom, or with a different functionalgroup. Accordingly, the term polypeptide includes amino acidic polymerof any length including full length proteins, and peptides as well asanalogs and fragments thereof. A polypeptide of three or more aminoacids is also called a protein oligomer or oligopeptide.

An “α-helix” or an “alpha-helix” is a form of regular secondarystructure of proteins that is right-hand-coiled or spiral confirmationin which every backbone N—H group donates a hydrogen bond to thebackbone C═O of the amino acid four residues earlier.

A “β-sheet”, a “beta-sheet”, a “β-pleated sheet” or a “beta-pleatedsheet” is a form of regular secondary structure of proteins. β-sheetsconsist of a plurality of β-strands connected laterally by at least twoor three backbone hydrogen bonds, forming a generally twisted, pleatedsheet. Accordingly, a “β-sheet” or a “beta-sheet”, is a stretch ofpolypeptide chain typically 3 to 10 amino acids long with backbone in anextended conformation.

A “β-ribbon”, a “beta-ribbon”, a “β-hairpin, a “beta hairpin”, a “β-βunit” or a “beta-beta unit” is a simple protein structural motifinvolving two β-strands that look like a hairpin. The motif consists oftwo strands that are adjacent in primary structure, oriented in ananti-parallel direction (the N-terminus of one sheet is adjacent to theC-terminus of the next), and linked by a short loop of two to five aminoacids. Beta hairpins can occur in isolation or as part of a series ofhydrogen bonded strands that collectively comprise a β-sheet.

FMDV 3C protease. The FMDV 3C protease is a 213-amino acid, 23.1-kDacysteine protease whose amino acid sequence is greater than 95%homologous across all known serotypes and strains of the virus. Thecysteine-histidine-aspartic acid catalytic triad at the active site ofthe FMDV 3C protease, which is conserved in all cysteine proteases, isformed by the residues H46, D84 and C163. Structurally, the FMDV 3Cprotease adopts a chymotrypsin-like fold that consists of the N-terminusn-barrel domain and the C-terminus (3-barrel domain (see FIG. 2). Eachof the n-barrel domains is composed of a pair of four-strandedanti-parallel β-sheets that pack together to form a peptide or substratebinding cleft. The β-sheets are composed of: B₁, A₁, D₁, E₁ β-strandsand B₁, C₁, F₁, E₁ β-strands for the N-terminus domain; and B₂, A₂, D₂,E₂ β-strands and B₂, C₂, F₂, E₂ β-strands for the C-terminus domain,where the B and E strands of each domain contributing to both β-sheets.Other secondary structures of the N-terminus and C-terminus n-barreldomains, in addition to the aforementioned β-strands, are loops or turnsconnecting the β-strands which include A₁-B₁, A₂-B₂, B₁-C₁, B₂-C₂,C₁-D₁, C₂-D₂, E₂-F₂ loops as non-limiting examples and the N-terminus,C-terminus α-helices, which are designated in FIG. 3 as α_(N), α_(C),respectively). FIG. 3 aligns the secondary structures of the FMDV 3Cprotease with their corresponding residues in the amino acid sequence.

In addition to the two n-barrel domains, the FMDV 3C protease possessesanother prominent tertiary structure in the form of a β-ribbon having asmall n-sheet of two short anti-parallel β-strands and an apical loopconnecting the two β-strands. As seen in FIG. 2, the β-ribbon folds overthe substrate binding cleft and active site and contributes to substraterecognition and specificity. The β-ribbon including the two β-strandsand the apical loop is formed by residues 138 to 150 of the FMDV 3Cprotease, as indicated in FIG. 3.

FMDV P1 precursor polypeptide (or P1 precursor protein) is a polypeptidecomprised of the FMDV structural proteins and/or precursors, VP0, VP1,VP2, VP3, and VP4, as well as the 2A translational interrupter. The FMDVP1 precursor is around 85 kDa in molecular weight. The P1 precursor isprocessed by the FMDV 3C protease into structural proteins forming VLPsand the FMDV capsid.

The FMDV VP0 protein is a precursor peptide comprised of the FMDV VP2and VP4 structural proteins. The FMDV VP0 protein is also identified asthe FMDV 1AB protein and is around 33 kDa in molecular weight. It isproduced by the processing of the FMDV P1 precursor protein by the FMDV3C protease. The FMDV VP0 protein is important in the formation ofprotomers along with FMDV proteins VP3 and VP1. Five of these protomersassemble into a pentamer and twelve pentamers can assemble into a FMDVcapsid or VLP. Cleavage of VP0 into VP2 and VP4 occurs through anunknown mechanism.

The FMDV VP1 protein is a structural protein which comprises the FMDVcapsid and/or FMDV VLP. The FMDV VP1 protein is also identified as theFMDV 1D protein and is around 24 kDa in molecular weight. The FMDV VP1protein contains a mobile loop structure, identified as the G-H loop,which emerges from the surface of the FMDV capsid and/or VLP. The FMDVVP1 protein can form a protomer along with VP0 and VP3. Five of theseprotomers assemble into a pentamer and twelve pentamers can assembleinto a FMDV capsid or VLP.

The FMDV VP2 protein is a structural protein which comprises the FMDVcapsid and/or FMDVVLP. The FMDV VP2 protein is also identified as theFMDV 1B protein and is around 24 kDa in molecular weight. The FMDV VP2protein, along with the FMDV VP4 protein, is part of the FMDV VP0protein until the formation of FMDV capsids and/or VLPs at which pointthe VP0 protein is processed into VP2 and VP4.

The FMDV VP3 protein is a structural protein which comprises the FMDVcapsid and/or FMDV VLP. The FMDV VP3 protein is also identified as theFMDV 1C protein and is around 24 kDa in molecular weight. The FMDV VP3protein can form a protomer along with VP0 and VP1. Five of theseprotomers assemble into a pentamer and twelve pentamers can assembleinto a FMDV capsid or VLP.

The FMDV VP4 protein is the smallest of the FMDV structural proteins andis part of the FMDV capsid and/or FMDV VLP. The FMDV VP4 protein is alsoidentified as the FMDV 1D protein and is around 9 kDa in molecularweight. The FMDV VP4 protein, along with the FMDV VP2 protein, is partof the FMDV VP0 protein until the formation of FMDV capsids and/or VLPsat which point the VP0 protein is processed into VP2 and VP4. Unlikeother FMDV proteins which comprise the capsid and/or VLP the VP4 proteinis entirely located inside the capsid and/or VLP structure.

“Virus-like particles” or “VLPs” resemble viruses, but arenon-infectious because they do not contain any viral genetic material.The expression of viral structural proteins, such as envelope or capsid,can result in the self-assembly of VLPs that can stimulate an immuneresponse in a mammalian organism. In other words, VLPs are often emptyviral envelopes or empty viral capsids that are capable of stimulatingan immune response like a full virus. Methods and problems associatedwith the production of VLPs in alternative systems include thosedescribed by Lee, et al., J. Biomed. Sci. 16:69 (Aug. 11, 2009),Srinivas, et al., Biologicals 44:64-68 (2016), Mayr, et al., Vaccine 19:2152-2162 (2001) and Niborski, et al., Vaccine 24: 7204-7213 (2006)which are each incorporated by reference.

2A is an FMDV translation interrupter sequence, see Luke, et al,Biotech. Genetic Eng. Revs. 26:223-260 (2009) which is incorporated byreferences. A 2A polynucleotide sequence is described by nucleotides34-87 of SEQ ID NO: 119 and by the amino acid residues encoded thereby.Other 2A sequences may conform to the amino acid motif described by SEQID NO: 193. 2A interrupters from other Apthoviruses may also be used.

Δ1D2A. A translation termination sequence that comprises FMDV 1Dresidues and the FMDV 2A amino acid residues. The polynucleotidesequence of SEQ ID NO: 119 encodes the Δ1D2A polypeptide of SEQ ID NO:120. Other degenerate sequences encoding the polypeptide of SEQ ID NO:120 may also be used. Polynucleotides 1-33 encode FMDV 1D residues,34-87 encode the 2A amino acid residues, and 88-90 encode a C-terminalproline residue described by both SEQ ID NOS: 119 and 120. Othertranslation termination sequences similar to Δ1D2A may have fewer ormore residues of the 1D protein than Δ1D2A or may contain 1, 2, 3 ormore point mutations to the 2A sequence that do not affect is ability toact as a translation termination sequence. Δ1D2A will comprise the aminoacid sequence PGP and may optionally comprise part or all of one of thefollowing motifs:

(SEQ ID NO: 206) (H/R/Y/D)(K/R)(Q/T/F/V)(E/K/P/A/D)(I/P/L/A)(I/T/V)(A/K/G/S)(P/V)(E/A/V)(K/R)Q(V/L/M/T)(L/C)(N/S) FDLLKLAGDVESNPGP.(SEQ ID NO: 207) (A/V/I/L/M/T)(T/S/L/C)(N/S)(F/K)(D/S/E)LL(K/Q/L)(Q/R/L)AGD(V/I)E(T/C/S)NPGP (SEQ ID NO: 208) AGD(V/I)E(T/C/S)NPGP(SEQ ID NO: 211) LLXXAGDXEXNPGP (SEQ ID NO: 212) DXEXNPGP

GLuc. Gaussia luciferase gene (GLuc) or a protein expressed therefrom,including variants that are luciferous, such as proteinshaving, with atleast 90, 95, or 99% sequence identity with a native GLuc protein. GLucis a small, naturally secreted luciferase of 185 amino acids (SEQ ID NO:201). GLuc has a higher intensity when compared to firefly or Renillaluciferases.

SGLuc. A Gaussia luciferase gene (GLuc) gene that expresses a secretoryfrom of the luciferase or the SGLuc luciferase that can be secreted. Amutation of amino acids 89 and 90 in GLuc produces a super luminescentGLuc variant (SGLuc, SEQ ID NO: 203) useful for examination of lowlevels of protein expression.

Quaternary FMDV Protein structures. The FMDV VP1, VP0, and VP3 proteinscan form a protomer structure consisting of a single copy of eachprotein. Five of these protomers, consisting of VP1, VP0, and VP3, canassemble into a pentamer structure.

Enzymes/Proteases

The term “enzyme” as used herein refers to any substance that catalyzesor promotes one or more chemical or biochemical reactions, which usuallyincludes enzymes totally or partially composed of a polypeptide, but caninclude enzymes composed of a different molecule includingpolynucleotides. A protease (also called a peptidase or proteinase) isany enzyme that performs proteolysis.

“Cysteine proteases” are also known as thiol proteases. These proteasedegrade proteins via a catalytic mechanism that involves a nucleophiliccysteine in a catalytic triad or dyad. The FMDV 3C is a cysteineprotease.

A “catalytic triad” or a “dyad” refers to the three amino acid residuesthat function at the center of an active site of some enzymes includingproteases, amidases, acylases, lipases and β-lactamases, as an acid, abase and a nucleophile respectively. These three residues form acharge-relay network to polarize and activate the nucleophile, whichattacks the substrate to form a covalent intermediate which is thenhydrolyzed to regenerate free enzyme. The nucleophile is most commonly aserine or cysteine amino acid, but occasionally threonine.

FMDV 3C protease activity. The 3C protease cleaves the FMDV precursorprotein at the positions shown in FIG. 7 to produce viral proteins VP0,VP3 and VP1. Other activities of the native 3C protease includesuppression of host cell protein production, processing of host cellproteins, fragmentation of the Golgi apparatus, induction of the loss ofmicrotubule system integrity, and induction of the loss of gamma-tubulinfrom the microtubule organizing center.

Prophylaxis/Treatment

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, prevention delays disease onset, reduces severity, reducescontagion, or otherwise alters disease symptoms and presentation.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. cattle) is cured and the disease iseradicated. Rather, embodiments of the present disclosure alsocontemplate treatment that delays disease progression, decreasesparticular symptoms, reduces contagion, or otherwise affects diseasepresentation or symptoms or progression. Prevention or treatment with avaccine according to the invention may involve the induction of cellular(e.g., via T-cells) or humoral (e.g., via antibodies) immunity. Such avaccine will usually contain one or more FMDV antigens produced by ahost cell expressing a modified FMDV 3C protease. However, DNA-basedvaccines that express FMDV 3C protease and other FMDV antigen(s) arealso contemplated. The term “in vivo” referring to a reaction, such asbut not limited to production of FMDV virus-like particles, geneexpression (e.g. an FMDV polypeptide precursor, a wild-type or modifiedFMDV 3C protease, etc.), DNA transcription, mRNA translation, cleavingof an FMDV polypeptide precursor (e.g. P1, etc.), means that thereaction takes place within the environment of a living cell, such as aviral host cell. The living cell may be a living cell inside a host orother organism or in an artificial culture medium.

The term “in vitro” referring to a reaction, such as but not limited toproduction of FMDV virus-like particles, gene expression (e.g. an FMDVpolypeptide precursor, a wild-type or modified FMDV 3C protease, etc.),DNA transcription, mRNA translation, cleaving of an FMDV polypeptideprecursor (e.g. P1, etc.), means that the reaction takes place in anyenvironment with the exception of a living cell, including not limitedto a solution, a liquid/solid culture medium in a test tube, a flask, apetri dish, etc.

Sequence Homology/Identity/Similarity

The term “homolog” used with respect to an original enzyme or gene of afirst family or species, refers to distinct enzymes or genes of a secondfamily or species which are determined by functional, structural orgenomic analyses to be an enzyme or gene of the second family or specieswhich corresponds to the original enzyme or gene of the first family orspecies. Most often, homologs will have functional, structural orgenomic similarities. Techniques are known by which homologs of anenzyme or gene can readily be cloned using genetic probes and PCR.Identity of cloned sequences as homolog can be confirmed usingfunctional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if theamino acid sequence encoded by a gene has a similar amino acid sequenceto that of the second gene. Alternatively, a protein has homology to asecond protein if the two proteins have “similar” amino acid sequences.Thus, the term “homologous proteins” is defined to mean that the twoproteins have similar amino acid sequences.

A mutant, variant or modified polypeptide may have 75, 80, 85, 90, 95,97.5, 98, 99, or 100% sequence identity or sequence similarity with aknown FMDV polynucleotide or polypeptide sequence, such as thosedescribed herein and in the sequence listing.

BLASTN may be used to identify a polynucleotide sequence having at least70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequenceidentity to a reference polynucleotide. A representative BLASTN settingoptimized to find highly similar sequences uses an Expect Threshold of10 and a Wordsize of 28, max matches in query range of 0, match/mismatchscores of 1/-2, and linear gap cost. Low complexity regions may befiltered or masked. Default settings of a Standard Nucleotide BLAST aredescribed by and incorporated by reference toblast.ncbi.nlm.nih.gov/_Blast.cgi?PROGRAM=blastn&BLASTPROGRAMS=megaBlast& PAGE_TYPE=BlastSearch&SHOW DEFAULTS=on&LINKLOC=blasthome (last accessed Feb. 4, 2016).

BLASTP can be used to identify an amino acid sequence having at least70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequenceidentity, or similarity to a reference amino acid using a similaritymatrix such as BLOSUM45, BLOSUM62 or BLOSUM80 where BLOSUM45 can be usedfor closely related sequences, BLOSUM62 for midrange sequences, andBLOSUM80 for more distantly related sequences. Unless otherwiseindicated a similarity score will be based on use of BLOSUM62. WhenBLASTP is used, the percent similarity is based on the BLASTP positivesscore and the percent sequence identity is based on the BLASTPidentities score. BLASTP “Identities” shows the number and fraction oftotal residues in the high scoring sequence pairs which are identical;and BLASTP “Positives” shows the number and fraction of residues forwhich the alignment scores have positive values and which are similar toeach other. Amino acid sequences having these degrees of identity orsimilarity or any intermediate degree of identity or similarity to theamino acid sequences disclosed herein are contemplated and encompassedby this disclosure. A representative BLASTP setting that uses an ExpectThreshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and GapPenalty of 11 (Existence) and 1 (Extension) and a conditionalcompositional score matrix adjustment. Other default settings for BLASTPare described by and incorporated by reference to the disclosureavailable at:blast.ncbi.nlm.nih.gov/_Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome (last accessed Jun. 29, 2016).

Techniques

A “polymerase chain reaction”, or a “PCR”, is a laboratory techniqueused to make multiple copies of a segment of DNA. PCR can be used toamplify, or copy, a specific DNA target from a mixture of DNA/mRNA/cDNAmolecules. First, two short DNA/RNA sequences called primers aredesigned to bind to the start and end of the DNA target. Then, toperform PCR, the DNA/mRNA/cDNA template that contains the target isadded to a tube that contains primers, free nucleotides, and an enzymecalled DNA polymerase, and the mixture is placed in a PCR machine. ThePCR machine increases and decreases the temperature of the sample inautomatic, programmed steps. Initially, the mixture is heated todenature, or separate, the double-stranded DNA/mRNA/cDNA template intosingle strands. The mixture is then cooled so that the primers anneal,or bind, to the DNA template. At this point, the DNA polymerase beginsto synthesize new strands of DNA starting from the primers. Followingsynthesis and at the end of the first cycle, each double-stranded DNAmolecule consists of one new and one old DNA strand. PCR then continueswith additional cycles that repeat the aforementioned steps. The newlysynthesized DNA segments serve as templates in later cycles, which allowthe DNA target to be exponentially amplified millions of times. A PCRcan also be used to introduce one or more mutations and types ofmutations to amplified copies of a DNA segment, such as but not limitedto a site directed mutagenesis PCR.

The foot-and-mouth disease virus (FMDV) is a non-enveloped picornavirus(belonging to the genus Aphthovirus of the family Picornaviridae) with asingle-stranded genomic RNA of between 7,500 to 8,000 nucleotides orapproximately between 7,500 to 8,000 nucleotides, approximately 7,500nucleotides, or approximately 8,000 nucleotides. Referring to FIG. 1,the FMDV RNA genome is translated in a single open reading frame as asingle polypeptide precursor which must be cleaved into functionalproteins by virally encoded proteases. Such cleavages take place atdifferent stages as shown in FIG. 1, forming multiple intermediatepolypeptide precursors and yielding the final protein products of capsidstructural proteins VP1, VP2, VP3 and VP4, as well as non-structuralproteins L, 2A, 2B, 2C, 3A, 3B₁, 3B₂, 3B₃, 3C and 3D.

The most important of the virally encoded proteases is the 3C proteasewhich is responsible for more than half of the required peptidecleavages, including the cleavage of the P1 polypeptide precursor intothe capsid proteins VP0, VP3, and VP1 during capsid assembly. The FMDVcapsid or protein shell is formed by assembly of 60 copies of each ofthe four structural proteins VP1-VP4.

Generating a strong protective immune response after vaccination againstFMDV is associated with the delivery or assembly of FMDV capsids in thehost. In principle, empty capsids produced by a molecular or DNAvaccine, which are non-infectious, can result in the self-assembly ofrecombinant virus-like particles (VLPs). However, the formation ofstable VLPs in host cells at concentrations high enough to stimulateimmune responses are severely hindered by the FMDV 3C protease becausethe proteolytic activity of the enzyme is not limited to processing ofthe viral peptides. It has been shown that FMDV 3C protease is able toinduce proteolytic cleavage of several host proteins including histoneH3, nuclear transcription factor kappa B essential modulator (NEMO),Src-associated substrate in mitosis of 68 kDa (SAM68), eukaryotictranslation initiation factor 4A1 (eIF4A1), and eukaryotic translationinitiation factor 4G (eIF4G). The presence of the 3C protease has alsobeen shown to induce Golgi fragmentation and loss of microtubule systemintegrity of a host cell.

The present invention is based, in part, on modifications to anucleotide sequence encoding a foot-and-mouth disease virus (FMDV) 3Cprotease that reduce or eliminate the toxicity of the expressed proteasetowards a host cell, compared to the wild-type FMDV 3C protease.Preferably and advantageously, these altered proteases retain theirability to fully process and cleave an FMDV P1 polypeptide precursorinto individual capsid proteins VP1, VP2 VP3 and VP4 or VP0, VP1 and VP3to allow subsequent assembly of these cleaved viral capsid proteins intoan FMDV empty capsid in the host cell.

The inventors provide herein multiple mutagenic strategies for reducingor eliminating the toxicity of an FMDV 3C protease towards a host cell,while retaining the ability of the FMDV 3C protease to at leastpartially, preferably completely, process and cleave the P1 polypeptideprecursor to form individual FMDV capsid proteins VP1, VP2, VP3 and VP4or VP0, VP1 and VP3. Such non-limiting strategies include one or morepoint mutations to a nucleotide sequence encoding an FMDV 3C proteasethat would result in one or more amino acid or residue substitutions inthe translated amino acid sequence, specifically one or more pointmutations targeting one or more of A₁-F₁ and A₂-F₂ β-strands of the twoβ-barrel domains. Accordingly, a non-limiting example of a mutant FMDV3C protease provided herein contains 1-12 amino acid substitutions inits amino acid sequence, as compared to the wild-type FMDV 3C protease,preferably 1-10, 1-8, 1-6 or 1-5 amino acid substitutions, morepreferably 1-4 or 1-3 amino acid substitutions, most preferably 1 or 2amino acid substitutions.

The modified 3C protease of the invention processes FMDV P1 precursoreffectively while exhibiting reduced toxicity of host cells. Thisproteolytic activity as measured by cleavage of P1 at one or more sitesmay be more or less than a corresponding non-modified or native 3Cprotease. The modified 3C protease of the invention, as measured by theproduction of cleaved P1 precursor, may exhibit 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150 or 200% of the proteolyticactivity of a corresponding non-modified 3C protease. In onenon-limiting example, the modified FMDV 3C protease exhibits at least90% of the ability to cleave P1 compared to a corresponding unmodifiedor native 3C protease. This activity may be determined based on therelative ability of the modified 3C protease to perform one or morecleavages of P1, such as those producing VP0, VP1 and VP3 viralproteins, or in another embodiment or VP1, VP2, VP3 and VP4.

In other embodiments, the modified FMDV 3C protease may exhibit lessthan 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10% of the proteolyticactivity of the corresponding unmodified protease on one or more hostcell proteins at one or more host protein target sites or on otherco-expressed proteins, such as other polypeptide components of amultivalent vaccine while retaining a significant ability to processFMDV P1 precursor protein. In some embodiments, the modified FMDV 3Cprotease exhibits no higher than 10% proteolytic activity towards a hostprotein, including, but not limited to the eIF4A1 translation factor.

In other embodiment, the growth rate of a host cell or the yield of atleast one FMDV antigen for a transformed host cell will be increased byat least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 300% or more for a host cell in which the modified FMDV 3C proteaseis expressed compared to a host cell expressing the correspondingunmodified FMDV 3C protease.

In yet another embodiment, the viability or passage stability of a hostcell (the ability of the host cell to stably maintain and express frompassage-to-passage a nucleic acid encoding at least one FMDV antigen)expressing the modified FMDV 3C protease may be increased by 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 200% or more compared to an otherwiseidentical host cell expressing the corresponding unmodified FMDV 3Cprotease.

The 3C polynucleotide sequence may be modified so as to introduce anamino acid substitution at position 127 of the 3C protease, such as aL127P substitution (i.e., the substitution of proline for leucine atposition 127). A polynucleotide sequence encoding the 3C protease mayalso be modified so as to encode amino acid deletions, substitutions oradditions at the positions corresponding to positions 138 to 150 of awild-type 3C protease. These residues of the wild-type 3C protease formthe β-ribbon of the protease, for example, such a modifiedpolynucleotide sequence can encode a C142T substitution.

A wild-type FMDV protease polynucleotide sequence may be obtained fromany of the seven major FMDV serotypes of O, A, C, Asia 1, SAT1, SAT2 andSAT3 or from any other wild-type FMDV. The same or similar modificationsmay be made to synthetic or engineered 3C polynucleotide sequences or tonatural or engineered mutant 3C polynucleotide sequences. Moreover, the3C polynucleotide sequences of attenuated, modified, or engineered FMDVstrains may be further modified to contain the one or more nucleotidepoint mutations described above or the other modifications describedherein.

Further modifications may be made to a polynucleotide sequence encodinga modified 3C protease. For example, prior to the transformation of ahost cell, codon frequency of a polynucleotide sequence encoding FMDV 3Cprotease, or other FMDV antigens may be modified to optimize expressionor stability of a nucleic acid encoding FMDV 3C protease, or other FMDVantigens. Software suitable for optimizing codon usage is known and maybe used to optimize codon usage in nucleic acid encoding FMDV 3Cprotease, or other FMDV antigens, see Optimizer: A web server utilitythat optimize a DNA or Protein sequence, created by P. Puigbo,Evolutionary Genomics Group, Biochemistry and Biotechnology DepartmentUniversitat Rovira i Virgili (URV), Tarragona, Spain. Codon usagefrequencies for various organisms are known and are also incorporated byreference to the Codon Usage Database, Kazusa DNA Research Institute,Chiba, Japan.

Not all amino acid codons are degenerate, for example, in the geneticcode of most organisms, Met and Trp are encoded by single codons.However, for degenerate codons, frequency or average frequency of codonusage may be selected to range from 0% (no common degenerate codons) to100% (same frequency of codon usage as host cell genome). This rangeincludes all intermediate values include 0%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% and 100%. Similarly, G+C content of a nucleicacid encoding FMDV 3C protease or other FMDV antigens may be matched,moved closer or moved away from that of the host cell by selection of adegenerate codon with more or fewer G or C nucleotides. G+C content ofexogenous nucleic acids encoding FMDV 3C protease or other FMDV antigensmay range within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50% more orless than the average G+C content of the host cell.

Alternatively, codon usage may be modified to modulate or control theexpression of FMDV 3C protease or other FMDV antigens or to attenuatethe expression of host cell proteins required for host cell viability,growth, or robustness; see for example, Kew, et al., U.S. Pat. No.8,846,051 hereby incorporated by reference. In some embodiments,expression of FMDV 3C or FMDV antigens by a host cell may be limited orreduced by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or more comparedto a maximum expression rate (e.g., where codon frequency is matched tothe host cell) in order to permit aggregation of an antigen (e.g., intoa quaternary structure) or a particular folding of an expressed protein(e.g., having a particular secondary or tertiary structure).

A vector may carry polynucleotides encoding modified FMDV 3C proteasesequences and/or encoding FMDV P1 polypeptide precursors. The vectorinduces production of FMDV virus-like particles in a host cell whenexpressed in the host cell. In certain embodiments, the vector has atotal transgene expression output in the host cell that is up to 20times higher, relative to a vector expressing at least a wild-type FMDV3C protease and the FMDV P1 polypeptide precursor in a host cell. Infurther embodiments, the vector has a total FMDV virus-like particleproduction in the host cell that is up to 20 times higher, relative to avector expressing at least a wild-type FMDV 3C protease and the FMDV P1polypeptide precursor in a host cell.

In one embodiment, the modified FMDV 3C protease and the FMDV P1polypeptide precursor may be expressed in a single open reading frame orin separate reading frames.

A vector may further comprise a nucleotide sequence encoding atranslation regulatory element. In one particular embodiment, thetranslation regulatory element is a translational interrupter sequenceand the nucleotide sequence encoding therefor is positioned between theengineered nucleotide sequence encoding an engineered FMDV 3C proteaseand the nucleotide sequence encoding an FMDV P1 polypeptide precursor toallow the engineered FMDV 3C protease and the FMDV P1 polypeptideprecursor to be independently expressed.

Polynucleotide sequences may be spliced or otherwise inserted intovectors or polynucleotide constructs which may further include apromoter sequence, a nucleotide sequence for initiation of eukaryotictranslation, an enhancer sequence, or other sequence to facilitate orotherwise modulate or control expression of the FMDV 3C protease orother FMDV antigen.

A vector may be a mammalian expression vector. However, those of skillin the art will be able to select a suitable vector for expression of anFMDV antigen in a particular host cell. For example, a vector suitablefor transformation and protein expression in an insect cell can be usedto express FMDV antigens in an insect cell line such as Sf9, Sf21 orHigh Five insect cells. Other expression systems for FMDV 3C protease orFMDV antigens include those for yeast, such as Saccharomyces cerevisiaeor Pichia pastoris, filamentous fungi, such as Aspergillus, Trichoderma,and Myceliophthora thermophila, non-lytic insect cell systems, such asSf9, Sf21 from Spodoptera frugiperda cells, Hi-5 from Trichoplusia nicells, and Schneider 2 cells and Schneider 3 cells from Drosophilamelanogaster; plant expression systems, including tobacco, and otherhuman or mammalian expressions systems, for example, from mice, rats,rabbits, hamsters, bovines, porcine, etc. Vectors or DNA constructs forexpression of FMDV antigens in prokaryotes may also be used, such asthose useful for expression in E. coli, Bacillus, Corynebacterium, orPseudomonas. A minicircle or other kind of vector may be used to expressa modified 3C protease.

An embodiment of the polynucleotide sequence encoding a modified 3Cprotease may also be engineered to place the structural genes encodingthe 3C protease or other FMDV antigens under the control of one or moreinducible promoter(s) to regulate the level of expression of the 3Cprotease or other FMDV antigens. For example, such promoters may beselected to optimize the concentrations of 3C protease and P1 precursorpolypeptide and reduce exposure of a host cell to high levels of FMDVcomponents which may slow host cell growth or its ability to expressexogenous polypeptides. Such promoters are known and include thoseregulated by tetracycline, steroids, metals, alcohol and other organiccompounds. The use of different promoters to express the protease andother FMDV antigens permits the control and optimization of the relativeamounts of the FMDV 3C protease and other FMDV antigens, such as P1protein precursor. For example, one may minimize the effects of an FMDV3C protease on the host cell, by limiting the amount expressed to onlythat necessary to process the P1 protein precursor.

A host cell transformed or transfected with a vector encoding a modified3C protease polynucleotide sequence and a FMDV P1 polypeptide precursorsequence may exhibit increased expression of FMDV antigens compared toan otherwise identical vector expressing a native 3C protease due toreduced toxicity of a modified 3C protease on host cells. Totaltransgene expression by a host cell expressing a modified 3C proteaseaccording to the invention can be increased by at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more timescompared to a host cell expressing the native 3C protease.

A host cell transformed or transfected with a vector encoding a modified3C protease polynucleotide sequence and a FMDV P1 polypeptide precursorsequence also may exhibit increased production of FMDV virus-likeparticles, compared to an otherwise identical vector expressing a native3C protease. Total FMDV virus-like particle formation by a host cellexpressing a modified 3C protease according to the invention can beincreased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 or more times compared to a host cell expressing anative 3C protease.

An embodiment of the polynucleotide sequence encoding a modified 3Cprotease may be further modified to contain one or more other ancillarypolynucleotide sequences that facilitate intracellular processing ortrafficking of FMDV antigens expressed by a host cell, theiraggregation, for example into VLPs, their secretion from a cell, ortheir identification, quantification or recovery, e.g. by co-expressionof luciferase or other biological tags.

In further embodiments, a mutant FMDV 3C protease provided herein hasthe ability to process at least 90% of the total amount of an FMDV P1polypeptide precursor expressed in a host cell, preferably at least 95%,more preferably at least 99%, even more preferably at least 99.9%, mostpreferably 99.9-100.0%.

In other embodiments, a mutant FMDV 3C protease provided herein has theability to process at least 90% of the total amount of an FMDVpolypeptide precursor other than the FMDV P1 polypeptide precursor. TheFMDV polypeptide precursor other than the FMDV P1 polypeptide precursormay include the single FMDV polypeptide precursor translation product,and intermediate FMDV polypeptide precursors P2 (or 2ABC), P3 (or3ABCD), 1ABCD, 1ABC, 2BC, 3AB, and 3CD. Modified or mutant FMDV 3Cproteases according to the invention may be used to cleave or processnative sites in native FMDV proteins or the same or similar 3C cleavagesites in non-FMDV proteins, such as proteins engineered to include FMDV3C cleavage sites.

In some embodiments, a mutant FMDV 3C protease described herein willhave decreased proteolytic activity toward the eIF4A1 eukaryoticinitiation factor compared to an otherwise identical unmodified FMDV 3Cprotease. A mutant FMDV 3C protease may degrade only 0.001, 0.01, 0.1,1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95% of an eIF4A1 eukaryoticinitiation factor compared to an otherwise identical unmodified FMDV 3Cprotease. Preferably such decreased activity is no higher than 10% basedon the total amount of eIF4A1 expressed in a host cell, preferably nohigher than 8%, more preferably no higher than 5%, even more preferablyno higher than 1%, and most preferably 0.001-1.0%.

A modified FMDV 3C protease may exhibit a loss of activity towards atleast one of histone H3, nuclear transcription factor kappa B essentialmodulator (NEMO), Src-associated substrate in mitosis of 68 kDa (SAM68)and/or eukaryotic translation initiation factor 4G (eIF4G). The modifiedprotease may degrade only 0.001, 0.01, 0.1, 1, 5, 10, 20, 30, 40, 50,60, 70, 80, 90 or 95% of at least one of histone H3, nucleartranscription factor kappa B essential modulator (NEMO), Src-associatedsubstrate in mitosis of 68 kDa (SAM68) and/or eukaryotic translationinitiation factor 4G (eIF4G) compared to an otherwise identicalunmodified FMDV 3C protease In further embodiments, the ability of theFMDV 3C protease to induce Golgi fragmentation and loss of microtubulesystem integrity of a host cell is reduced or eliminated by the one ormore mutations of the present disclosure.

Modification of the FMDV 3C protease may increase the expression outputof a transgene expression cassette or a recombinant expression vectorcontaining at least a mutant nucleotide sequence encoding a mutant FMDV3C protease and a nucleotide sequence encoding an FMDV P1 polypeptideprecursor. In some non-limiting embodiments, the transgene expressionoutput is increased by up to 20 times, preferably 2-20 times, morepreferably 5-15 times, even more preferably 10-15 times. When a hostcell is transfected with a transgene expression cassette or arecombinant expression vector containing at least a mutant nucleotidesequence encoding a mutant FMDV 3C protease and a nucleotide sequenceencoding an FMDV P1 polypeptide precursor, the increase in the transgeneexpression output would translate into an increase in the production ofFMDV virus-like particles (VLPs) in a host cell.

In one embodiment, the transgene expression output is assessed by fusinga luminescent reporter gene to the transgene expression cassette, suchas a Gaussia luciferase gene (GLuc) or a variant thereof including, butnot limited to SGLuc and then measuring the number of relative lightunits (RLU) utilizing an integration time of 0.5 seconds on aluminometer. In some non-limiting embodiments, a recombinant expressionvector containing a transgene expression cassette or a recombinantexpression vector containing at least a mutant nucleotide sequenceencoding a mutant FMDV 3C protease and a nucleotide sequence encoding anFMDV P1 polypeptide precursor has a transgene expression output in ahost cell of 10⁹-10¹⁰ RLU/0.5 s, preferably 2×10⁹ to 8×10¹⁰ RLU/0.5 s,and more preferably 4×10⁹ to 3×10¹⁰ RLU/0.5 s.

Mutations resulting in amino acid substitutions may be introduced into amutant FMDV 3C protease of the invention using any methodology known tothose skilled in the art. Mutations may be introduced randomly by, forexample, conducting a PCR reaction in the presence of manganese as adivalent metal ion cofactor. In another embodiment, oligonucleotidedirected mutagenesis may be used to create the modified FMDV 3Cproteases which allows for all possible classes of base pair changes atany determined site along the encoding DNA molecule. In general, thistechnique involves annealing an oligonucleotide complementary (exceptfor one or more mismatches) to a single stranded nucleotide sequencecoding for the FMDV 3C protease of interest. The mismatchedoligonucleotide is then extended by DNA polymerase, generating adouble-stranded DNA molecule which contains the desired amino acidsubstitution in sequence in one strand. The double-strandedpolynucleotide can then be inserted into an appropriate expressionvector, and a mutant or modified polypeptide can thus be produced. Theabove-described oligonucleotide directed mutagenesis or site directedmutagenesis can, optionally, be carried out via PCR.

The nucleotide sequence encoding an FMDV 3C protease may be derived fromany of the A, O, C, Asia 1, SAT1, SAT2 and SAT3 serotypes, as well asthe subtypes, topotypes and strains within these seven serotypes. Insome embodiments, the nucleotide sequence encoding an FMDV 3C proteaseincludes but is not limited to SEQ ID NO: 1 (Asia Lebanon 89, serotypeAsia 1), SEQ ID NO: 3 (O1 Manisa isolate 87 strain, serotype O), SEQ IDNO: 5 (O1 PanAsia), SEQ ID NO: 7 (A24 Cruzeiro iso71), SEQ ID NO: 9 (ATurkey 2006), SEQ ID NO: 11 (SAT2 Egypt 2010), SEQ ID NO: 13 (C3Indaial), SEQ ID NO: 15 (SAT3 ZIM/6/91), SEQ ID NO: 17 (SAT1-20 iso11),and SEQ ID NO: 19 (Asial Shamir). SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18 and 20 respectively describe the proteins encoded by SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17 and 19.

One embodiment of the invention is a modified FMDV 3C protease that hasbeen modified on a surface region that is distal from the substratebinding cleft and the proteolytic active site of the FMDV 3C protease.This embodiment involves the modification of the 3C protease in one ormore domains, regions or segments distinct from sites known toparticipate in substrate recognition and proteolysis for the purpose ofreducing toxicity of the 3C protease while maintaining its proteolyticactivity on FMDV P1 precursor protein. To the surprise of the inventors,such modifications outside of known active sites had significant andadvantageous effects on the specificity of the 3C protease.

In another embodiment the invention is directed to a polynucleotide orpolynucleotide construct, such as a chimeric polynucleotide orexpression vector, that encodes a polypeptide that is at least 70%, 80%,90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 and that proteolyticallycleaves foot-and-mouth disease virus (FMDV) P1 polypeptide, wherein saidpolypeptide contains one or more deletions, additions or substitutionsin the positions corresponding to residues 28, 125 to 134, 142 and 163of a polypeptide of SEQ ID NO: 2, 4, 6, 8. 10, 12, 14, 16, 18 or 20;wherein said protease exhibits decreased host cell toxicity whenexpressed in a host cell and/or increases the yield of at least one FMDVantigen when co-expressed with one or more polynucleotide sequencesencoding FMDV antigen(s) in a host cell, compared to an otherwiseidentical polypeptide that does not contain the one more deletions,additions or substitutions in the positions corresponding to residues26-35, 125-134 or 138-150, in some embodiments residues 28, 125 to 134,142 and 163, of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20.

Alternatively, said encoded polypeptide may differ from the amino acidsequence encoded by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 by1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50 ormore amino acid deletions, substitutions or insertions as long as itexhibits decreased host cell toxicity when expressed in a host celland/or increases the yield of at least one FMDV antigen or viral proteinwhen co-expressed with one or more polynucleotide sequences encodingFMDV antigen(s) in a host cell compared to an otherwise identicalpolypeptide that does not contain the one more deletions, additions orsubstitutions in the positions corresponding to residues 28, 125 to 134,142 and 163 of a native 3C protein.

Fragments of said encoded polypeptide, or hybrid or chimeric proteinscontaining such fragments, that differ from a polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 by at least one deletion,substitution or addition of an amino acid residue are also contemplatedas long as they retain the ability to proteolytically cleave FMDV P1polypeptide and exhibit decreased host cell toxicity when expressed in ahost cell and/or increases the yield of at least one FMDV antigen in ahost cell, compared to an otherwise identical polypeptide fragment thatdoes not contain the one more deletions, additions or substitutions inthe positions corresponding to residues 26-35, 125-134 or 138-150 of apolypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20.

In some embodiments, it is preferred that one or more point mutationsare introduced to a region of the nucleotide sequence encoding an FMDV3C protease, specifically a region encoding the B₂ β-strand of the FMDV3C protease to result in one or more amino acid substitutions in thecorresponding amino acid sequence forming the B₂ β-strand. The B₂β-strand of the FMDV 3C protease is formed by residues 125 to 134 (10residues) and therefore has a consensus amino acid sequence ofGRLIFSG(D/E)AL (SEQ ID NO: 156).

Referring to FIG. 2, the 10 residues forming the B₂ β-strand of the FMDV3C protease are surface residues of the protein molecule and are each atleast 3.5 Å, preferably 5.0-20.0 Å, and more preferably 7.5-15.0 Å awayfrom the substrate binding cleft and the active site of the protease asmeasured between a B₂ β-strand residue and any of the catalytic triadresidues (i.e., H46, D84 and C163) and are therefore incapable offorming hydrogen bond or other chemical interactions with any residue atthe substrate binding cleft or the active site. It is thereforeunexpected that one or more amino acid substitutions in the B₂ β-strandwould result in a mutant FMDV 3C protease that retains the ability tofully process the P1 polypeptide while dramatically enhancing transgeneoutput and does not or minimally induces cytotoxicity to the host cell.

Preferably, one or more amino acids from residues 127 to 134 aremodified. Residues 125 to 134 have a consensus amino acid sequence ofGRLIFSG(D/E)AL (SEQ ID NO: 156). Any one of the residues 125 to 134 maybe modified by another amino acid, where each amino acid modification isresulted by at least one nucleotide change in the codon encoding theresidue. As a non-limiting example, glycine at position 125 (G125) maybe substituted with an alanine, an arginine, an asparagine, an asparticacid, a cysteine, a glutamine, a glutamic acid, a histidine, anisoleucine, a leucine, a lysine, a methionine, a phenylalanine, aproline, a serine, a threonine, a tryptophan, a tyrosine, a valine, oran unnatural amino acid; arginine at position 126 may be substitutedwith an alanine, an asparagine, an aspartic acid, a cysteine, aglutamine, a glutamic acid, a glycine, a histidine, an isoleucine, aleucine, a lysine, a methionine, a phenylalanine, a proline, a serine, athreonine, a tryptophan, a tyrosine, a valine, or an unnatural aminoacid; leucine at position 127 (L127) may be substituted with an alanine,an arginine, an asparagine, an aspartic acid, a cysteine, a glutamine, aglutamic acid, a glycine, a histidine, an isoleucine, a lysine, amethionine, a phenylalanine, a proline, a serine, a threonine, atryptophan, a tyrosine, a valine, or an unnatural amino acid. Isoleucineat position 128 (I128) may be substituted with alanine, an arginine, anasparagine, an aspartic acid, a cysteine, a glutamine, a glutamic acid,a glycine, a histidine, a leucine, a lysine, a methionine, aphenylalanine, a proline, a serine, a threonine, a tryptophan, atyrosine, a valine, or an unnatural amino acid. Phenylalanine atposition 129 (F129) may be substituted with an alanine, an arginine, anasparagine, an aspartic acid, a cysteine, a glutamine, a glutamic acid,a glycine, a histidine, an isoleucine, a leucine, a lysine, amethionine, a proline, a serine, a threonine, a tryptophan, a tyrosine,a valine, or an unnatural amino acid. Glycine at position 130 (G130) maybe substituted with an alanine, an arginine, an asparagine, an asparticacid, a cysteine, a glutamine, a glutamic acid, a histidine, anisoleucine, a leucine, a lysine, a methionine, a phenylalanine, aproline, a serine, a threonine, a tryptophan, a tyrosine, a valine, oran unnatural amino acid. Aspartic acid or glutamic acid at position 131(D131 or E131) may be substituted with an alanine, an arginine, anasparagine, a cysteine, a glutamine, a glutamic acid (for D131) or anaspartic acid (for E131), a histidine, an isoleucine, a leucine, alysine, a methionine, a phenylalanine, a proline, a serine, a threonine,a tryptophan, a tyrosine, a valine, or an unnatural amino acid. Alanineat position 132 (A132) may be substituted with an arginine, anasparagine, an aspartic acid, a cysteine, a glutamine, a glutamic acid,a glycine, a histidine, an isoleucine, a leucine, a lysine, amethionine, a phenylalanine, a proline, a serine, a threonine, atryptophan, a tyrosine, a valine, or an unnatural amino acid. Leucine atposition 130 (L130), like L127, may be substituted with an alanine, anarginine, an asparagine, an aspartic acid, a cysteine, a glutamine, aglutamic acid, a glycine, a histidine, an isoleucine, a lysine, amethionine, a phenylalanine, a proline, a serine, a threonine, atryptophan, a tyrosine, a valine, or an unnatural amino acid.

More preferably, one or more amino acids from residues 127 to 130 (4residues) are modified. Residues 127 to 130 have a consensus amino acidsequence of LIFS. As a non-limiting example, any one or more of theresidues 127 to 130 may be substituted by another amino acid, where eachamino acid substitution is resulted by at least one nucleotide change inthe codon encoding the residue. For example, leucine at position 127(L127) may be substituted with an alanine, an arginine, an asparagine,an aspartic acid, a cysteine, a glutamine, a glutamic acid, a glycine, ahistidine, an isoleucine, a lysine, a methionine, a phenylalanine, aproline, a serine, a threonine, a tryptophan, a tyrosine, a valine, oran unnatural amino acid. Isoleucine at position 128 (I128) may besubstituted with an alanine, an arginine, an asparagine, an asparticacid, a cysteine, a glutamine, a glutamic acid, a glycine, a histidine,a leucine, a lysine, a methionine, a phenylalanine, a proline, a serine,a threonine, a tryptophan, a tyrosine, a valine, or an unnatural aminoacid. Phenylalanine at position 129 (F129) may be substituted with analanine, an arginine, an asparagine, an aspartic acid, a cysteine, aglutamine, a glutamic acid, a glycine, a histidine, an isoleucine, aleucine, a lysine, a methionine, a proline, a serine, a threonine, atryptophan, a tyrosine, a valine, or an unnatural amino acid. Glycine atposition 130 (G130) may be substituted with an alanine, an arginine, anasparagine, an aspartic acid, a cysteine, a glutamine, a glutamic acid,a histidine, an isoleucine, a leucine, a lysine, a methionine, aphenylalanine, a proline, a serine, a threonine, a tryptophan, atyrosine, a valine, or an unnatural amino acid.

Most preferably, a modified FMDV 3C protease provided herein has theleucine residue at position 127 (L127) modified. As a non-limitingexample, L127 may be substituted with an alanine, an arginine, anasparagine, an aspartic acid, a cysteine, a glutamine, a glutamic acid,a glycine, a histidine, an isoleucine, a lysine, a methionine, aphenylalanine, a proline, a serine, a threonine, a tryptophan, atyrosine, a valine, or an unnatural amino acid, which is resulted by atleast one nucleotide change in the codon encoding the L127 residue.Preferably, L127 is substituted with an alanine, an asparagine, anaspartic acid, a cysteine, a glutamine, a glutamic acid, a glycine, ahistidine, a lysine, a phenylalanine, a proline, a serine, a threonine,a tryptophan and a tyrosine.

In one embodiment, the L127 of an FMDV 3C protease is substituted with aproline. The L127P substitution is resulted by any one of the followingnucleotide changes in a leucine codon:TTA/TTG/CTT/CTC/CTA/CTG→CCT/CCC/CCA/CCG. In one embodiment, the L127Psubstitution is resulted by a single nucleotide change in CTG to CCG.

In a further embodiment, a mutant nucleotide sequence encoding a L127Pmutant FMDV 3C protease is selected from but not limited to:

SEQ ID NO: 21 (L127P Asia Lebanon 89, serotype Asia 1),

SEQ ID NO: 23 (L127P O1 Manisa isolate 87 strain, serotype O),

SEQ ID NO: 25 (L127P O1 PanAsia),

SEQ ID NO: 27 (L127P A24 Cruzeiro iso71),

SEQ ID NO: 29 (L127P A Turkey/2006),

SEQ ID NO: 31 (L127P SAT2 Egypt 2010),

SEQ ID NO: 33 (L127P C3 Indaial),

SEQ ID NO: 35 (L127P SAT3 ZIM/6/91),

SEQ ID NO: 37 (L127P SAT1-20 iso11), and

SEQ ID NO: 39 (L127P Asial Shamir).

In another embodiment, the amino acid sequence of the L127P mutant FMDV3C protease is selected from but not limited to:

SEQ ID NO: 22 (L127P Asia Lebanon 89, serotype Asia 1),

SEQ ID NO: 24 (L127P O1 Manisa isolate 87 strain, serotype O),

SEQ ID NO: 26 (L127P O1 PanAsia),

SEQ ID NO: 28 (L127P A24 Cruzeiro iso71),

SEQ ID NO: 30 (L127P A Turkey/2006),

SEQ ID NO: 32 (L127P SAT2 Egypt 2010),

SEQ ID NO: 34 (L127P C3 Indaial),

SEQ ID NO: 36 (L127P SAT3 ZIM/6/91),

SEQ ID NO: 38 (L127P SAT1-20 iso11), and

SEQ ID NO: 40 (L127P Asial Shamir).

In certain embodiments, it is preferred that the arginine residue atposition 126 (R126) is not subjected to any amino acid modification. Inother words, R126 is not substituted with an alanine, an asparagine, anaspartic acid, a cysteine, a glutamine, a glutamic acid, a glycine, ahistidine, an isoleucine, a leucine, a lysine, a methionine, aphenylalanine, a proline, a serine, a threonine, a tryptophan, atyrosine, a valine, or an unnatural amino acid. As a non-limitingexample, a mutant FMDV 3C protease provided herein does not include asubstitution of R126 with an acidic amino acid, such as aspartic acidand glutamic acid. More particularly, the mutant FMDV 3C protease doesnot include a substitution of R126 with a glutamic acid (i.e., R126E).

In certain embodiments, it is preferred that the alanine at position 133(A133) is not subjected to any amino acid modification. In other words,A133 is not substituted with an arginine, an asparagine, an asparticacid, a cysteine, a glutamine, a glutamic acid, a glycine, a histidine,an isoleucine, a leucine, a lysine, a methionine, a phenylalanine, aproline, a serine, a threonine, a tryptophan, a tyrosine, a valine, oran unnatural amino acid. As a non-limiting example, a mutant FMDV 3Cprotease provided herein does not include a substitution of A133 with aserine (i.e., A133S).

In some embodiments, one or more point mutations are introduced, insteadof or in addition to one or more regions of the nucleotide sequenceencoding an FMDV 3C protease other than the region encoding the B₂β-strand. In certain embodiments, one or more point mutations areintroduced, instead of or in addition to a region encoding the β-ribbonof the FMDV 3C protease to result in one or more amino acidsubstitutions in the corresponding amino acid sequence forming theβ-ribbon, and/or a region encoding the B₁ β-strand of the FMDV 3Cprotease to result in one or more amino acid substitutions in thecorresponding amino acid sequence forming the B₁ β-strand. As shown inFIG. 3, the β-ribbon of the FMDV 3C protease is formed by residues 138to 150 (13 residues) and has an amino acid sequence of D(I/L)VVCMDGDTMPF(SEQ ID NO: 157). The B₁ β-strand of the FMDV 3C protease is formed byresidues 26 to 35 (10 residues) and has an amino acid sequence ofKTVA(I/L)CCATF (SEQ ID NO: 158).

Preferably, one or more amino acids from residues 140 to 143 (4residues) and/or residues 27 to 30 (4 residues) are modified. Residues140 to 143 have an amino acid sequence of VVCM (SEQ ID NO: 159) whileresidues 27 to 30 have an amino acid sequence of TVAI (SEQ ID NO: 160).Any one of the residues 140 to 143 and 27 to 30 may be substituted byanother amino acid, where each amino acid substitution is resulted by atleast one nucleotide change in the codon encoding the residue. As anon-limiting example, valine at position 140 (V140) may be substitutedwith an alanine, an arginine, an asparagine, an aspartic acid, acysteine, a glutamine, a glutamic acid, a glycine, a histidine, anisoleucine, a leucine, a lysine, a methionine, a phenylalanine, aproline, a serine, a threonine, a tryptophan, a tyrosine, or anunnatural amino acid. Valine at position 141 (V141), like V140, may besubstituted with an alanine, an arginine, an asparagine, an asparticacid, a cysteine, a glutamine, a glutamic acid, a glycine, a histidine,an isoleucine, a leucine, a lysine, a methionine, a phenylalanine, aproline, a serine, a threonine, a tryptophan, a tyrosine, or anunnatural amino acid. Cysteine at position 142 (C142) may be substitutedwith an alanine, an arginine, an asparagine, an aspartic acid, aglutamine, a glutamic acid, a glycine, a histidine, an isoleucine, aleucine, a lysine, a methionine, a phenylalanine, a proline, a serine, athreonine, a tryptophan, a tyrosine, a valine, or an unnatural aminoacid. Methionine at position 143 (M143) may be substituted with analanine, an arginine, an asparagine, an aspartic acid, a cysteine, aglutamine, a glutamic acid, a glycine, a histidine, an isoleucine, aleucine, a lysine, a phenylalanine, a proline, a serine, a threonine, atryptophan, a tyrosine, a valine, or an unnatural amino acid. Threonineat position 27 (T27) may be substituted with an alanine, an arginine, anasparagine, an aspartic acid, a cysteine, a glutamine, a glutamic acid,a glycine, a histidine, an isoleucine, a leucine, a lysine, amethionine, a phenylalanine, a proline, a serine, a tryptophan, atyrosine, a valine, or an unnatural amino acid. Valine at position 28(V28), like V140 and V141, may be substituted with an alanine, anarginine, an asparagine, an aspartic acid, a cysteine, a glutamine, aglutamic acid, a glycine, a histidine, an isoleucine, a leucine, alysine, a methionine, a phenylalanine, a proline, a serine, a threonine,a tryptophan, a tyrosine, or an unnatural amino acid. Alanine atposition 29 (A29) may be substituted with an arginine, an asparagine, anaspartic acid, a cysteine, a glutamine, a glutamic acid, a glycine, ahistidine, an isoleucine, a leucine, a lysine, a methionine, aphenylalanine, a proline, a serine, a threonine, a tryptophan, atyrosine, a valine, or an unnatural amino acid. Isoleucine at position30 (I30) may be substituted with alanine, an arginine, an asparagine, anaspartic acid, a cysteine, a glutamine, a glutamic acid, a glycine, ahistidine, a leucine, a lysine, a methionine, a phenylalanine, aproline, a serine, a threonine, a tryptophan, a tyrosine, a valine, oran unnatural amino acid.

More preferably, one or more amino acids from residues at positions 141and 142 (V141, C142) and/or residue at position 28 (V28) are modified.Any one of the residues V141, C142 and V28 may be substituted by anotheramino acid, where each amino acid substitution is resulted by at leastone nucleotide change in the codon encoding the residue. As anon-limiting example, valine at position 141 (V141) may be substitutedwith an alanine, an arginine, an asparagine, an aspartic acid, acysteine, a glutamine, a glutamic acid, a glycine, a histidine, anisoleucine, a leucine, a lysine, a methionine, a phenylalanine, aproline, a serine, a threonine, a tryptophan, a tyrosine, or anunnatural amino acid. Cysteine at position 142 (C142) may be substitutedwith an alanine, an arginine, an asparagine, an aspartic acid, aglutamine, a glutamic acid, a glycine, a histidine, an isoleucine, aleucine, a lysine, a methionine, a phenylalanine, a proline, a serine, athreonine, a tryptophan, a tyrosine, a valine, or an unnatural aminoacid. Valine at position 28 (V28), like V141, may be substituted with analanine, an arginine, an asparagine, an aspartic acid, a cysteine, aglutamine, a glutamic acid, a glycine, a histidine, an isoleucine, aleucine, a lysine, a methionine, a phenylalanine, a proline, a serine, athreonine, a tryptophan, a tyrosine, or an unnatural amino acid.

In one embodiment, a mutant FMDV 3C protease in accordance with thepresent invention contains a single amino acid modification where theresidue at position 28 (V28) is substituted with a lysine. The V28Ksubstitution is resulted by any one of the following nucleotide changesin a leucine codon: GTT/GTC/GTA/GTG→AAA/AAG). In one embodiment, theV28K substitution is resulted by a nucleotide change of GTG to AAG.

In a further embodiment, a mutant nucleotide sequence encoding a V28Kmutant FMDV 3C protease is selected from but not limited to SEQ ID NO:41 (V28K Asia Lebanon 89, serotype Asia 1),

SEQ ID NO: 43 (V28K 01 Manisa isolate 87 strain, serotype O),

SEQ ID NO: 45 (V28K 01 PanAsia),

SEQ ID NO: 47 (V28K A24 Cruzeiro iso71),

SEQ ID NO: 49 (V28K A Turkey/2006),

SEQ ID NO: 51 (V28K SAT2 Egypt 2010),

SEQ ID NO: 53 (V28K C3 Indaial),

SEQ ID NO: 55 (V28K SAT3 ZIM/6/91),

SEQ ID NO: 57 (V28K SAT1-20 iso11), and

SEQ ID NO: 59 (V28K Asial Shamir).

In another embodiment, the amino acid sequence of the V28K mutant FMDV3C protease is selected from but not limited to:

SEQ ID NO: 42 (V28K Asia Lebanon 89, serotype Asia 1),

SEQ ID NO: 44 (V28K 01 Manisa isolate 87 strain, serotype O),

SEQ ID NO: 46 (V28K 01 PanAsia),

SEQ ID NO: 48 (V28K A24 Cruzeiro iso71),

SEQ ID NO: 50 (V28K A Turkey/2006),

SEQ ID NO: 52 (V28K SAT2 Egypt 2010),

SEQ ID NO: 54 (V28K C3 Indaial),

SEQ ID NO: 56 (V28K SAT3 ZIM/6/91),

SEQ ID NO: 58 (V28K SAT1-20 iso11), and

SEQ ID NO: 60 (V28K Asial Shamir).

In one embodiment, a mutant FMDV 3C protease in accordance with thepresent invention contains a single amino acid substitution where theresidue at position 141 (V141) is substituted with a lysine. The V141Tsubstitution is resulted by any one of the following nucleotide changesin a leucine codon: GTT/GTC/GTA/GTG→ACT/ACC/ACA/ACG). In one embodiment,the V141T substitution is resulted by a nucleotide change of GTG to ACG.

In a further embodiment, a mutant nucleotide sequence encoding a V141Tmutant FMDV 3C protease is selected from but not limited to:

SEQ ID NO: 61 (V141T Asia Lebanon 89, serotype Asia 1),

SEQ ID NO: 63 (V141T O1 Manisa isolate 87 strain, serotype O),

SEQ ID NO: 65 (V141T O1 PanAsia),

SEQ ID NO: 67 (V141T A24 Cruzeiro iso71),

SEQ ID NO: 69 (V141T A Turkey/2006),

SEQ ID NO: 71 (V141T SAT2 Egypt 2010),

SEQ ID NO: 73 (V141T C3 Indaial),

SEQ ID NO: 75 (V141T SAT3 ZIM/6/91),

SEQ ID NO: 77 (V141T SAT1-20 iso11),

SEQ ID NO: 79 (V141T Asial Shamir).

In another embodiment, the amino acid sequence of the V141T mutant FMDV3C protease is selected from but not limited to:

SEQ ID NO: 62 (V141T Asia Lebanon 89, serotype Asia 1),

SEQ ID NO: 64 (V141T O1 Manisa isolate 87 strain, serotype O),

SEQ ID NO: 66 (V141T O1 PanAsia),

SEQ ID NO: 68 (V141T A24 Cruzeiro iso71),

SEQ ID NO: 70 (V141T A Turkey/2006),

SEQ ID NO: 72 (V141T SAT2 Egypt 2010),

SEQ ID NO: 74 (V141T C3 Indaial),

SEQ ID NO: 76 (V141T SAT3 ZIM/6/91),

SEQ ID NO: 78 (V141T SAT1-20 iso11),

SEQ ID NO: 80 (V141T Asial Shamir).

Most preferably, the cysteine residue at position 142 (C142) ismodified, instead of or in addition to an amino acid substitution at theleucine residue at position 127 (L127). As a non-limiting example,cysteine at position 142 (C142) may be substituted with an alanine, anarginine, an asparagine, an aspartic acid, a glutamine, a glutamic acid,a glycine, a histidine, an isoleucine, a leucine, a lysine, amethionine, a phenylalanine, a proline, a serine, a threonine, atryptophan, a tyrosine, a valine, or an unnatural amino acid, which isresulted by at least one nucleotide change in the codon encoding theC142 residue. L127 may be substituted with an alanine, an arginine, anasparagine, an aspartic acid, a cysteine, a glutamine, a glutamic acid,a glycine, a histidine, an isoleucine, a lysine, a methionine, aphenylalanine, a proline, a serine, a threonine, a tryptophan, atyrosine, a valine, or an unnatural amino acid, which is resulted by atleast one nucleotide change in the codon encoding the L127 residue.

In one embodiment, a mutant FMDV 3C protease is a double mutant proteasewhere residues at positions 127 (L127) and 142 (C142) have beensubstituted. As a non-limiting example, leucine at position 127 (L127)may be substituted with an alanine, an arginine, an asparagine, anaspartic acid, a cysteine, a glutamine, a glutamic acid, a glycine, ahistidine, an isoleucine, a lysine, a methionine, a phenylalanine, aproline, a serine, a threonine, a tryptophan, a tyrosine, a valine, oran unnatural amino acid. Preferably, L127 is substituted with analanine, an asparagine, an aspartic acid, a cysteine, a glutamine, aglutamic acid, a glycine, a histidine, a lysine, a phenylalanine, aproline, a serine, a threonine, a tryptophan and a tyrosine. Cysteine atposition 142 (C142) may be substituted with an alanine, an arginine, anasparagine, an aspartic acid, a glutamine, a glutamic acid, a glycine, ahistidine, an isoleucine, a leucine, a lysine, a methionine, aphenylalanine, a proline, a serine, a threonine, a tryptophan, atyrosine, a valine, or an unnatural amino acid. Preferably, C142 issubstituted with an alanine, a histidine, an isoleucine, a leucine, amethionine, a phenylalanine, a serine, a threonine or a tyrosine. Morepreferably, C142 is substituted with an alanine, a leucine, a serine ora threonine.

In one embodiment, a mutant FMDV 3C protease is a double mutant proteasehaving the amino acid substitutions L127P and C142T. The L127Psubstitution is resulted by any one of the following nucleotide changesin a leucine codon: TTA/TTG/CTT/CTC/CTA/CTG→CCT/CCC/CCA/CCG). In oneembodiment, the L127P substitution is resulted by a single nucleotidechange in CTG to CCG. The C142T substitution is resulted by any one ofthe following nucleotide changes in a cysteine codon:TGT/TGC→ACT/ACC/ACA/ACG. In one embodiment, the C142T substitution isresulted by a nucleotide change of TGC to ACA.

In a further embodiment, a mutant nucleotide sequence encoding aL127P/C142T double mutant FMDV 3C protease is selected from but notlimited to:

SEQ ID NO: 81 (L127P/C142T Asia Lebanon 89, serotype Asia 1),

SEQ ID NO: 83 (L127P/C142T O1 Manisa isolate 87 strain, serotype O),

SEQ ID NO: 85 (L127P/C142T O1 PanAsia),

SEQ ID NO: 87 (L127P/C142T A24 Cruzeiro iso71),

SEQ ID NO: 89 (L127P/C142T A Turkey/2006),

SEQ ID NO: 91 (L127P/C142T SAT2 Egypt 2010),

SEQ ID NO: 93 (L127P/C142T C3 Indaial),

SEQ ID NO: 95 (L127P/C142T SAT3 ZIM/6/91),

SEQ ID NO: 97 (L127P/C142T SAT1-20 iso11),

SEQ ID NO: 99 (L127P/C142T Asial Shamir).

In another embodiment, the amino acid sequence of the L127P/C142T doublemutant FMDV 3C protease is selected from but not limited to:

SEQ ID NO: 82 (L127P/C142T Asia Lebanon 89, serotype Asia 1),

SEQ ID NO: 84 (L127P/C142T O1 Manisa isolate 87 strain, serotype O),

SEQ ID NO: 86 (L127P/C142T O1 PanAsia),

SEQ ID NO: 88 (L127P/C142T A24 Cruzeiro iso71),

SEQ ID NO: 90 (L127P/C142T A Turkey/2006),

SEQ ID NO: 92 (L127P/C142T SAT2 Egypt 2010),

SEQ ID NO: 94 (L127P/C142T C3 Indaial),

SEQ ID NO: 96 (L127P/C142T SAT3 ZIM/6/91),

SEQ ID NO: 98 (L127P/C142T SAT1-20 iso11),

SEQ ID NO: 100 (L127P/C142T Asial Shamir).

Transgene Expression Cassettes Comprising a Mutant Nucleotide SequenceEncoding a Mutant FMDV 3C Protease

Another aspect of the present invention is directed to a transgeneexpression cassette containing a mutant nucleotide sequence encoding amutant FMDV 3C protease as described herein. In one or more embodiments,the mutant nucleotide sequence is selected from those encoding a L127Pmutant of FMDV 3C (SEQ ID NOS: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39),a V28K mutant of FMDV 3C (SEQ ID NOS: 41, 43, 45, 47, 49, 51, 53, 55,57, 59), V141T (SEQ ID NOS: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79) anda L127P/C142T double mutant of FMDV 3C (SEQ ID NOS: 81, 83, 85, 87, 89,91, 93, 95, 97, 99).

The structural gene/nucleic acid encoding the 3C protein may containadditional deletions, substitutions or insertions besides thosedescribed by the SEQ ID NOS: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, for example, it may containmodifications to the polynucleotide sequence that alter the identitiesof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues in the FMDV3C amino acid sequence outside of or distal to the substrate bindingcleft of the catalytically active site of the FMDV 3C protease.Alternatively or additionally, such modifications to the polynucleotidesequence may alter the identities of amino acid residues in the twon-barrel domains or β-ribbon of the 3C protease or to residues in thesubstrate binding cleft or in the catalytic triad residues of H46, D84and C163.

In another embodiment, the transgene expression cassette furtherincludes a nucleotide sequence encoding the FMDV P1 polypeptideprecursor. The nucleotide sequence encoding the P1 polypeptide precursorcan be derived from any of the A, O, C, Asia 1, SAT1, SAT2 and SAT3serotypes, as well as the subtypes, topotypes and strains within theseseven serotypes or other FMDV isolates or variants. In some embodiments,the nucleotide sequence encoding an FMDV P1 polypeptide precursor isselected from but not limited to:

SEQ ID NO: 101 (O1 Manisa Iso87),

SEQ ID NO: 103 (O1 PanAsia),

SEQ ID NO: 105 (A24 Cruzeiro iso71),

SEQ ID NO: 107 (A Turkey/2006),

SEQ ID NO: 109 (SAT2 Egypt 2010),

SEQ ID NO: 111 (C3 Indaial),

SEQ ID NO: 113 (SAT3 ZIM/6/91),

SEQ ID NO: 115 (SAT1 KNP/196/91), and

SEQ ID NO: 117 (Asial Shamir).

The amino acid sequences of the P1 polypeptides encoded by thenucleotide sequences immediately above are described by SEQ ID NOS: 102,104, 106, 108, 110, 112, 114, 116 and 118. A P1 polypeptide may bemodified, for example, by deletion, substitution or insertion of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more amino acid residues providedthat it retains or contains one or more epitopes, including both humoraland cellular epitopes, recognized by an animal's immune system (e.g., aFMDV mammalian host). Native P1 polypeptides are at least 62-100%homologous. In some embodiments a P1 polypeptide according to theinvention may be described as one capable of processing and assemblyinto an FMDV capsid. A P1 polypeptide may also be structurally describedas having 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% (or anyintermediate value) identical or similar to any of the P1 polypeptidesdisclosed herein or to those known in the art.

In other embodiments, the nucleotide sequence encoding an FMDV P1polypeptide precursor that is modified to remove internal restrictionsites is selected from, but not limited to:

SEQ ID NO: 136 (O1 Manisa Iso87 restriction sites removed),

SEQ ID NO: 137 (O1 PanAsia restriction sites removed),

SEQ ID NO: 138 (A24 Cruzeiro iso71 restriction sites removed),

SEQ ID NO: 139 (A Turkey/2006 restriction sites removed),

SEQ ID NO: 140 (SAT2 Egypt 2010 restriction sites removed),

SEQ ID NO: 141 (C3 Indaial restriction sites removed),

SEQ ID NO: 142 (SAT3 ZIM/6/91 restriction sites removed),

SEQ ID NO: 143 (SAT1 KNP/196/91 restriction sites removed), and

SEQ ID NO: 144 (Asial Shamir restriction sites removed).

In one embodiment, the transgene expression cassette of the presentdisclosure comprises a nucleotide sequence encoding the P1 polypeptideprecursor that is derived from the 01 Manisa isolate 87 strain (SEQ IDNO: 101) and a mutant nucleotide sequence encoding the L127P mutant FMDVAsia Lebanon 89 protease of SEQ ID NO: 21. Such a transgene constructmay be cloned into a vector or polynucleotide construct and transfectedinto a host cell.

In another embodiment of the present invention, mutagenesis strategiesand techniques as described herein may be applied to introduce one ormore mutations to the nucleotide sequence encoding the polypeptideprecursor to enhance the stability of the final assembled capsidproduct. Among the mutations that can be introduced include, but are notlimited to nonsense mutations that effectively eliminate restrictionenzyme recognition sites to better facilitate cloning and sub-cloningyet maintain the same translated protein product by not causing anyamino acid substitution. These mutations enhance the cloning in andcloning out of the P1 polypeptide precursor into a transgene expressioncassette to swap different P1 polypeptide precursors from different FMDVserotypes to promptly respond to the needs of individual outbreaks.

In a further embodiment, the transgene expression cassette of thepresent disclosure further includes restriction enzyme recognition sitesor sequences at each of the N-terminus and C-terminus of the expressioncassette for cloning into an expression vector. Examples of theserestriction enzyme recognition sites include but are not limited torecognition sequences for EcoRI, EcoRII, BamHI, HindIII, Taql, Nod,HinFI, Sau3AI, PvuII, Smal, NheI, HaeIII, HgaI, AluI, EcoRV, KpnI, PstI,SacI, SpeI, StuI, SphI, XbaI, SalI, ScaI, XcmI, BsiWI, XhoI, BstEII,pilMI, AccI, SacII, PpuMI, AgeI, NcoI, BstXI, MluI and AatI.

In another embodiment, the transgene expression cassette of the presentdisclosure comprises a nucleotide sequence encoding the P1 polypeptideprecursor that is derived from the 01 Manisa isolate 87 strain (SEQ IDNO: 101) and a mutant nucleotide sequence encoding the L127P/C142Tmutant FMDV protease of SEQ ID NO: 81 from Asia Lebanon 89. Such atransgene construct may be cloned into a vector or polynucleotideconstruct and transfected into a host cell.

The transgene expression cassette described in accordance withembodiments described herein does not encode the complete FMDV genomeand therefore cannot cause an accidental FMD outbreak duringmanufacture, or administration of the vaccine containing the transgeneexpression cassette.

Furthermore, the transgene expression cassette encodes only one FMDVnon-structural protein, i.e. a mutant FMDV 3C protease. Animals treatedwith a vaccine containing the transgene expression cassette will notproduce antibodies to other FMDV non-structural proteins that areexpressed during a natural FMDV infection. For example, if the transgeneexpression cassette contains a mutant nucleotide sequence encoding amutant FMDV 3C protease it will only produce antibodies for the mutantFMDV 3C protease and not antibodies for other non-structural proteinssuch as 2B, 2C, 3B, 3D, etc. The difference in antibody profilesproduced after natural infection compared to vaccination with thetransgene expression cassette confers the ability to differentiateinfected animals from vaccinated animals and vice versa.

In one embodiment, the transgene expression cassette according to thedisclosure can be constructed as a single open reading frame. Thenucleotide sequence encoding the P1 polypeptide precursor may bepositioned 5′ or 3′ to the nucleotide sequence encoding a mutant FMDV 3Cprotease.

In certain embodiments, the transgene expression cassette furtherincludes a translational regulatory element that is located between thenucleotide sequence encoding a P1 polypeptide precursor and the mutantnucleotide sequence encoding a mutant FMDV 3C protease to advantageouslyallow for individual, equimolar expression of the two proteins in asingle open reading frame translation.

In some embodiments, the translational regulatory element is atranslational interrupter sequence of 5 to 50 amino acid residues long,preferably 15 to 40 residues, more preferably 25 to 35 residues. Infurther embodiments, the translational interrupter sequence can containportions of one or more FMDV non-structural proteins from any FMDVserotype (e.g., 1A, 1B, 1C, 1D, 2A, 2B, 2C, 3A, 3B, 3C, 3D). In oneparticular embodiment, the translational interrupter sequence is formedby incorporating an 11-amino acid of the C-terminus of the FMDV 1Dprotein to the 18-amino FMDV 2A protein and a proline residue to theC-terminus of the FMDV 2A protein, as shown in FIG. 4A.

In further embodiments, the nucleotide sequence encoding thetranslational interrupter sequence is SEQ ID NO: 119 and the amino acidsequence of the translational interrupter sequence is SEQ ID NO: 120. Inother embodiments the translation interrupter may comprise the motifdescribed by SEQ ID NO: 193 and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more non-2A residues such asC-terminal portions of FMDV 1D protein, or other portions of FMDV 1D, orFMDV viral proteins, or C-terminal portions of Aphthovirus 1D protein.

In some embodiments, the transgene expression cassette further includesa nucleotide sequence for initiation of translation in eukaryotes, suchas a Kozak consensus sequence. In one non-limiting embodiment, the Kozaksequence includes the nucleotide sequence of SEQ ID NO: 161. In anothernon-limiting embodiment, the Kozak eukaryotic translation initiationsequence includes the nucleotide sequence of SEQ ID NO: 162. In oneembodiment, the nucleotide sequence encoding the P1 polypeptideprecursor is positioned 5′ to the mutant nucleotide sequence encoding amutant FMDV 3C protease, and the eukaryotic translation initiationsequence is positioned upstream or 5′ to the nucleotide sequenceencoding the P1 polypeptide precursor. In an alternative embodiment, themutant nucleotide sequence encoding a mutant FMDV 3C protease ispositioned 5′ to the nucleotide sequence encoding the P1 polypeptideprecursor, and the eukaryotic translation initiation sequence ispositioned upstream or 5′ to the mutant nucleotide sequence encoding amutant FMDV 3C protease.

In further embodiments, the transgene expression cassette includes apromoter. Like the eukaryotic translation initiation sequence in certainembodiments, the promoter is positioned upstream or 5′ to the nucleotidesequence encoding the P1 polypeptide precursor or the mutant nucleotidesequence encoding a mutant FMDV 3C protease, depending on thearrangement of the two nucleotide sequences encoding the P1 polypeptideprecursor and the mutant FMDV 3C protease. In certain embodiments,strong and constitutive promoters such as but not limited to SV40, CMV,UBC, EF1A, PGK and CAG can be advantageously incorporated in thetransgene expression cassette for prolonged high levels of transgeneexpression in mammalian hosts to induce a strong immune response.

In further embodiments, a stop codon sequence (e.g., TAA, TGA, or TAG)may be added to the end of the transgene expression cassette to ensurecessation of mRNA translation.

In one embodiment, a transgene expression cassette in accordance withthe present disclosure is the “mpTarget O1P1-3C(L127P)” transgene thathas a nucleotide sequence of SEQ ID NO: 121. In yet another embodiment,a transgene expression cassette in accordance with the presentdisclosure is the L127P/C142T transgene “mpTarget O1P1-3CDNA” that has anucleotide sequence of SEQ ID NO: 122.

Recombinant Vectors Carrying the Transgene Expression Cassette

Another aspect of the present disclosure is directed to immunogeniccompositions, including one in the form of recombinant vectors orvehicles containing the transgene expression cassette as describedherein. Preferably, the transgene expression cassette is cloned into amammalian expression vector system. In one embodiment, the transgeneexpression cassette is cloned into a modified pTarget vector acquiredfrom Promega (mpTarget). In further embodiments, modifications to thepTarget vector include, but are limited to decreasing the overall vectorsize and/or removal of one or more restriction enzyme recognitionsequences at the multiple cloning site. In one embodiment, an emptymammalian expression vector “mpTarget Empty” that has a nucleotidesequence of SEQ ID NO: 123. In another embodiment, a mammalianexpression vector containing the transgene expression cassette insert“mpTarget O1P1-3CDNA” that has a nucleotide sequence of SEQ ID NO: 124.In yet another embodiment, a mammalian expression vector containing thetransgene expression cassette insert “mpTarget O1P1-3CDNA2” that has anucleotide sequence of SEQ ID NO: 125.

In one embodiment, the vector used for transferring the transgeneexpression cassette is a minicircle DNA vector. In a further embodiment,the minicircle DNA vector is a minicircle carrying a transgeneexpression cassette. In further embodiments, the minicircle DNA vectoris a minicircle carrying a transgene expression cassette and does notcontain an empty vector without an insert.

The use of a minicircle DNA vector to carry and transfer the transgeneexpression cassette allows mammalian cells to be transfected (e.g.,directly) without utilizing an intermediate eukaryotic host system(e.g., insect cell line production system). Directly transfecting amammalian cell with the minicircle DNA vector carrying the transgeneexpression cassette can eliminate the costs and labor associated withmaintaining large volumes of intermediate host cell cultures inproduction facilities and harvesting empty capsids or virus-likeparticle (VLPs) produced by intermediate host cells.

Furthermore, minicircle vectors are typically smaller than standardplasmid vectors and lack of extraneous bacterial sequences, both ofwhich enhance transfection of cells and enable an extended duration oftransgene expression within the mammalian host cell. For example, aminicircle vector is smaller than a standard vector as it lacksextraneous bacterial sequences found on plasmids. Differences in sizebetween plasmid vectors and minicircle vectors can be attributed to thelack of extraneous bacterial sequences, inclusion of an insubstantialamount of extraneous bacterial sequences in comparison to the overallsize of the vector, such as appreciably smaller in comparison to theplasmid, and variations thereof.

In one or more embodiments, an empty minicircle vector has a nucleotidesequence of SEQ ID NO: 126. In a further embodiment, the minicirclecontaining the transgene expression cassette “Minicircle O1P1-3CDNA”that has a nucleotide sequence of SEQ ID NO: 127. In yet a furtherembodiment, the minicircle containing the transgene expression cassette“Minicircle O1P1-3CDNA” that has a nucleotide sequence of SEQ ID NO:128.

Methods of producing minicircle vectors that are capable of inducingproduction of FMDV virus-like particles in mammalian host cells are alsoprovided herein. In one embodiment, minicircle vectors are preparedusing a two-step procedure. Firstly, a full-size parental plasmidcontaining bacterial sequences and transgene is produced in, forexample, Escherichia coli. While the parental plasmid is still insidethe E. coli host, the expression of a site-specific recombinase isinduced and the prokaryotic or bacterial bone is excised by the enzymeat the recombinase recognition sites. Non-limiting examples ofsite-specific recombinases include Tyr- and Ser-recombinases such as Crerecombinase, Flp recombinase, ParA resolvase and PhiC31 integrase. Anexample of suitable materials, techniques, approaches, and methods aredescribed in U.S. Pat. No. 8,236,548 which is hereby incorporated byreference in its entirety. Further description may be found in Kay etal, A Robust System for Production of Minicircle DNA Vectors, NatureBiotechnology, 28 1287-1289 (2010) which is hereby incorporated byreference in its entirety.

Transformed Host Cells

Another aspect of the present disclosure is directed to cells that aretransformed or transfected with a recombinant vector carrying atransgene expression cassette expressing at least an FMDV P1 polypeptideprecursor and a mutant FMDV 3C protease that is capable of fullyprocessing the FMDV P1 polypeptide precursor into individual FMDV capsidproteins of VP1, VP2, VP3 and VP4 or VP0, VP1 and VP3 without causingtoxicity to the transformed or transfected host cell. The host cells maybe prokaryotic, such as bacterial cells, or eukaryotic. Preferably, thehost cells are eukaryotic, such as but not limited to animal cells(particularly mammalian cells), plant cells and yeast cells. The hostcells may be transformed using any conventional transformationtechniques described herein. These cells may be grown under controlledconditions, generally outside of their natural environment prior toand/or post-transfection with the recombinant vector of the disclosure,such as in cell cultures, including but not limited to mammalian celllines and insect cell lines. In one embodiment, the human embryonickidney cell line HEK-293-T or the continuous porcine cell line LF-BKαV/β6 is used to host the recombinant vector carrying the transgeneexpression cassette. In an alternative embodiment, these cells are growninside of their natural environment, for example, as part of anorganism.

Foot-and-Mouth-Disease Virus Virus-Like Particles

Another aspect of the present disclosure is directed to FMDV virus-likeparticles (VLPs) and preparation methods thereof. VLPs are recombinantparticles with viral matrix or structural proteins such as capsids thatresemble viruses, but are non-infectious and unable to propagate asthey, respectively, do not contain any viral genetic material. VLPs canbe utilized as vaccine antigens as they mimic the native virions, andcan be produced in vitro in a variety of cell culture systems includingmammalian cell lines, insect cell lines, yeast and plant cells or invivo.

In certain embodiments of the present invention, an FMDV VLP is producedwhen an FMDV P1 polypeptide precursor is contacted with a mutant FMDV 3Cprotease as described herein. In one embodiment, the FMDV P1 polypeptideprecursor and the mutant FMDV 3C protease are co-expressed in vivoinside a host cell that is transformed with a recombinant vectorcarrying a transgene expression cassette containing at least a mutantnucleotide sequence encoding a mutant FMDV 3C protease and an FMDV P1polypeptide precursor, wherein the expressed FMDV P1 polypeptideprecursor is fully processed and cleaved by the expressed mutant FMDV 3Cinto individual FMDV capsid proteins of VP1, VP2, VP3 and VP4 or VP0,VP1 or VP3 and wherein these capsid proteins self-assemble to form FMDVVLPs, which are empty FMDV capsids. An FMDV VLP consists of one or moreassembled capsid proteins. These FMDV VLPs may be isolated and purifiedfrom their host cells.

In alternative embodiments, FMDV VLP production does not require aliving cell as a host cell, for example, when an FMDV P1 polypeptideprecursor is cleaved by a mutant FMDV 3C protease in vitro. In furtherembodiments, FMDV VLP production in vitro includes, but is not limitedto production in a test tube with appropriate solutions, buffers and/orcultural medium, or in a petri dish). The FMDV VLPs produced in this invitro manner may also be isolated and purified from their environments.

FMD Vaccine Compositions and Methods of Vaccinating a Subject in NeedThereof

Another aspect of the present disclosure is directed to compositions andimmunogenic preparations, including, but not limited to vaccinecompositions comprising the FMDV VLPs of the present disclosure. Incertain embodiments, the compositions and immunogenic preparations arecapable of inducing protective immunity in a suitably treated host and asuitable carrier. In other certain embodiments, the compositions andimmunogenic preparations are capable of inducing an immune response inthe form of specific antibody production or in cellular immunity wheninjected into a host.

A foot-and-mouth disease vaccine (FMD vaccine) or a foot-and-mouthdisease virus vaccine (FMDV vaccine) refers an immunogenic, biologicalcomposition that provides or improve immunity to one or more strains ofthe FMDV and to FMD. Such immunogenic compositions or vaccines areuseful, for example, in immunizing hosts against infection and/or damagecaused by the FMDV.

In certain embodiments, the vaccine preparations of the presentdisclosure can include an immunogenic amount of one or more FMDV VLPsisolated and purified from a host cell culture, fragment(s), orsubunit(s) thereof. In other embodiments, the vaccines can include oneor more FMDV capsid proteins and portions thereof, and adjuvant moleculeand portions thereof on the surfaces of the FMDV VLPs, or in combinationwith another protein or other immunogen, including, but not limited toone or more additional FMDV viral components naturally associated withviral particles or an epitopic peptide derived therefrom.

An “immunogenic amount” is an amount capable of eliciting the productionof antibodies directed against one or more strains of FMDV, in the hostto which an FMDV immunogenic composition or an FMD vaccine has beenadministered.

In an alternative embodiment, the vaccine preparations of the presentdisclosure can include an immunogenic amount of one or more recombinantvectors carrying a transgene expression cassette expressing at least anFMDV P1 polypeptide precursor and a mutant FMDV 3C protease that iscapable of fully processing the FMDV P1 polypeptide precursor intoindividual FMDV capsid proteins of VP1, VP2, VP3 and VP4 or VP0, VP1 andVP3 without causing toxicity to the transformed or transfected hostcell. In a further embodiment, a host, which includes, but is notlimited to a mammalian subject is protected against one or more strainsof the FMDV by injecting it with genetically engineered DNA (e.g.,transgene expression cassette+expression vector) to produce an immuneresponse through assembly of FMDV VLPs in situ in the host.

There are a number of advantages associated with DNA vaccine platforms,in comparison to traditional whole-pathogen vaccines and protein-basedvaccines. For example, DNA vaccines do not contain an actual infectiousagent, whether dead or alive. DNA vaccines can also be easilylyophilized for long-term storage and transportation and do not requireany cold chain delivery.

Additionally, the DNA vector inside a DNA vaccine can be produced andmodified more quickly and more easily in comparision to traditionalvaccine preparation. This allows a more rapid response to specificallyengineer DNA vaccines tailored to individual FMD outbreaks, including,but not limited to a DNA vaccine matching a specific FMDV outbreakstrain or serotype. In some embodiments, using a minicircle DNA vectorto carry and transfer the transgene expression cassette eliminates theuse of an intermediate eukaryotic host system and the associated costsand labor, including modification of an intermediate host system duringan outbreak, such as during the onset of an FMD outbreak.

In one or more embodiments, the immunogenic compositions and/or vaccinesof the present disclosure may be formulated by any of the methods knownin the art. They can be typically prepared as injectables (e.g.subcutaneous, intradermal and intramuscular injection, jet injections)or as formulations for oral administration, intranasal administration(e.g. aerosol inhalation or instillation), topical administration to theeye, electroporation, gene gun, transfection, liposome-mediated deliveryor combinations thereof, either as liquid solutions or suspensions.Solid forms suitable for solution in, or suspension in, liquid prior toinjection or other administration may also be prepared. The preparationmay also be emulsified or encapsulated in liposomes.

In one or more embodiments, the immunogenic compositions and/or vaccinesof the present disclosure may be formulated as multivalent or polyvalentvaccines containing immunogenic compositions that stimulate an immuneresponse towards two or more different strains of the same species or ofdifferent species. In a further embodiment, the multivalent vaccines ofthe present disclosure contain at least one FMDV VLP that is formed byFMDV capsid proteins processed by a mutant FMDV 3C protease of thepresent disclosure or a recombinant vector of the present disclosure. Ina further embodiment, the multivalent vaccines may include otherimmunogenic compositions, including, but not limited to full microbesthat are either live, killed, attenuated or inactivated; toxoidsthereof, subunits thereof; VLPs thereof; conjugates thereof; or nucleicacids thereof.

In one or more embodiments, the active immunogenic ingredients, such asthe FMDV VLP and recombinant vector, though not required, are oftenmixed with adjuvants, salts, carriers, excipients or diluents, which arepharmaceutically acceptable and compatible with the active ingredient.

In a further embodiment, adjuvants may be added to vaccine to modify theimmune response by boosting it such as to give a higher amount ofantibodies and a longer-lasting protection, thus minimizing the amountof injected foreign material. Adjuvants may also be used to enhance theefficacy of vaccine by helping to subvert the immune response toparticular cells type of immune system, for example by activating the Tcells instead of antibody-secreting B cells depending on the type of thevaccine. Example adjuvants include, but are not limited to,aqueous-based aluminum hydroxide gel-saponin, the oil-based MontanideISA 206, other aluminum-based adjuvants, incomplete Freunds adjuvant(IFA), and paraffin oil.

In a further embodiment, suitable excipients include but are not limitedto water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof.

In a further embodiment, example diluents include, but are not limitedto, water, physiological saline solution, human serum albumin, oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents, antibacterial agents such as benzyl alcohol, antioxidants suchas ascorbic acid or sodium bisulfate, chelating agents, such as ethylenediamine-tetra-acetic acid, buffers such as acetates, citrates orphosphates and agents for adjusting osmolarity, such as sodium chlorideor dextrose.

In a further embodiment, example carriers include, but are not limitedto, liquid carriers (e.g., water, saline, culture medium, saline,aqueous dextrose, aqueous glycols) and solid carriers (e.g.,carbohydrates such as starch, glucose, lactose, sucrose, dextrans;antioxidants such as ascorbic acid and glutathione, hydrolyzedproteins).

In a further embodiment, pharmaceutically acceptable salts, include butare not limited to, the acid addition salts (formed with free aminogroups of the peptide) which are formed with inorganic acids (e.g.,hydrochloric acid or phosphoric acids) and organic acids (e.g., acetic,oxalic, tartaric, or maleic acid). Salts formed with the free carboxylgroups may also be derived from inorganic bases (e.g., sodium,potassium, ammonium, calcium, or ferric hydroxides), and organic bases(e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine,and procaine).

In a further embodiment, the vaccines may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and/or other agents, which enhance the effectiveness of thevaccine. Examples of agents which may be effective include, but are notlimited to: aluminum hydroxide;N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP); N-acetyl muramyl-L-alanyl-D-isoglutaminy1-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE); and RIBI, which containsthree components extracted from bacteria: monophosphoryl lipid A,trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%squalene/Tween 80 emulsion. The effectiveness of the auxiliarysubstances may be determined by measuring the amount of antibodies(especially IgG, IgM or 19A) directed against the immunogen resultingfrom administration of the immunogen in vaccines which comprise theadjuvant in question. In a further embodiment, additional formulationsand modes of administration may also be used.

In one or more embodiments, the FMD vaccines formulated withcompositions and methods described herein may be administeredprophylactically (e.g., to prevent or ameliorate the effects of a futureinfection), therapeutically (e.g., to treat or to empower the immunesystem of an infected organism) or both, in a manner compatible with thedosage formulation, and in such an amount and manner as will beprophylactically and/or therapeutically effective. The quantity to beadministered for an FMDV VLP-based vaccine, is generally in the range of1-1000 μg, preferably 5-500 μg, more preferably 50-250 μg, even morepreferably 100-200 μg of pre-assembled FMDV VLPs per dose and/oradjuvant molecule per dose, depending on the subject to be treated, thecapacity of the host immune system to synthesize antibodies, and thedegree of protection desired. Precise amounts of the active ingredientrequired to be administered may depend on the judgment of theveterinarian or may be peculiar to each individual subject, but such adetermination is within the skill of such a practitioner.

In an alternative embodiment, if formulated as an FMD DNA vaccine inaccordance with embodiments of the present disclosure, the DNA vaccineis administered at dosages such as in the range of 25-1000 μg/μl of thea recombinant vector carrying the transgene expression cassette insaline solution, in the range of between 50-500 μg/μl, in the range of100-250 μg/μl. Other factors that can form the basis of what dosagerange to implement include but are not limited the size of the subject,how virulent the FMD strain that is being inoculated against is, and soforth factors that influence dosage amount. The FMD DNA vaccine and/orthe method of vaccinating a mammalian subject with the vaccine protectsthe subject against one or more of the 0, A, C, Asia 1, SAT1, SAT2 andSAT3 serotypes of the FMDV.

In a further embodiment, the vaccine or immunogenic composition may begiven in a single dose; two dose schedule, for example two to eightweeks apart; or a multiple dose schedule. A multiple dose schedule isone in which a primary course of vaccination may include 1 to 10 or moreseparate doses, followed by other doses administered at subsequent timeintervals as required to maintain and/or reinforce the immune response(e.g., at 1 to 4 10 months for a second dose, and if needed, asubsequent dose(s) after several months). Mammalian subjects immunizedwith the VLPs of the present disclosure are protected from infection bythe cognate virus.

In one or more embodiments, FMD vaccines in accordance with the presentdisclosure are marker vaccines or DIVA (Differentiating Infected fromVaccinated Animals), which induce immune responses that differ fromthose from natural infection. These differences are reflected inantibody profiles, and can be detected by diagnostic tests and assayssuch as enzyme linked immunosorbent assays (ELISAs) containing the samecompositions used in the vaccine formulations. The DIVA strategy isuseful in eradication scenarios wherein emergency vaccination using DIVAFMD vaccines could be an effective control tool for FMD outbreaks indensely populated livestock areas. DIVA vaccination can limit the numberof culled animals in the process of FMD eradication, thereby enhancingpublic acceptance for disease control measures and limiting economiclosses.

An FMD DNA vaccine's efficacy in embodiments is considered the rate ofreduction in the incidence of serotype-specific FMD among a populationof subjects that have been vaccinated compared to the incidence in apopulation of unvaccinated subjects, over a duration of 12 months.Vaccine efficacy can be measured using the following formula:VE=[(ARU−ARV)/ARU]×100%where “VE” is vaccine efficacy, “ARU” is an attack rate in anunvaccinated population and “ARV” is an attack rate in the vaccinatedpopulation.

In one or more embodiments, FMD DNA vaccines comprising the minicircleDNA vector in accordance with the present disclosure exhibit VE valuesof between 50-95%, approximately 50%, greater than 50%, 50%,approximately 75%, approximately 75%, greater than 75%, approximately90%, greater than 90%, 95%, approximately 95%, or greater than 95%.

It is to be understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a cell” includes a pluralityof such cells and reference to “the compound” includes reference to oneor more compounds and equivalents thereof known to those skilled in theart, and so forth. Numerous embodiments of the invention, many of whichinvolve the engineering of FMDV 3C protease variants, such as those thatare less toxic to host cells used to express FMDV viral proteins or VLPsbut which effectively process FMDV P1 protein, are disclosed.

One aspect of the invention is a polynucleotide that encodes a modifiedfoot-and-mouth disease (FMDV) 3C protease that comprises one or moreamino acid substitutions within residues 26-35, 125-134 or 138-150 of awild-type FMDV 3C protease. Generally, such a polynucleotide will encodea modified FMDV 3C protease that is at least 70, 75, 80, 85, 90, 95, 96,97, 98, or 99% similar or identical to at least one wild-type protease,such as a wild-type FMDV 3C protease from FMDV O serotype, A serotype, Cserotype, Asia 1 serotype, SAT1 serotype, SAT2 serotype, or SAT3serotype or that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99%similar or identical to at least one wild-type polynucleotide sequenceencoding a FMDV 3C protease selected from the group consisting of SEQ IDNOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 or that encodes a modifiedFMDV 3C protease that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or99% similar or identical to at least one wild-type FMDV 3C proteasedescribed by SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.

Such a polynucleotide sequence may also encode modified FMDV 3C proteasethat contains insertions, substitutions, or deletions of other aminoacid residues of a native 3C protease, or that retains native FMDV 3Cprotease residues. For example, a modified FMDV 3C protease may containone or more, or all, of the proline or cysteine residues of a nativeFMDV 3C protease or may contain substitutions or deletions of one ormore proline or cysteine residues of a native FMDV 3C protease. Forexample, the polynucleotide of the invention may encode a FMDV 3Cprotease that contains cysteine residues at positions 51 and 163 or thatencodes a modified FMDV 3C protease comprising residues H46, D84 andC163 of a native FMDV 3C protease.

In most embodiments, a polynucleotide of the invention will encode aFMDV 3C protease variant, or functionally active portions of such a 3Cprotease, that exhibit proteolytic activity on a FMDV P1 precursorpolypeptide or on other polypeptides containing FMDV 3C proteaserecognition sites. Such a polynucleotide will preferably encode amodified FMDV 3C protease that is less toxic to, less disruptive toprotein expression, less growth inhibitory to, or which exhibitsattenuated proteolytic activity against host cell proteins. However, insome embodiments, the variant FMDV 3C protease may have attenuated or noproteolytic activity against at least one or all of the FMDV 3C proteaserecognition sites in a FMDV P1 protein, but will exhibit other usefulproperties, such as antigenicity, immunogenicity, or an ability tomodulate cleavage or processing of FMDV P1 precursor protein or otherproteins recognized by a functionally active FMDV 3C protease.

A polynucleotide according to the invention may encode a FMDV 3Cprotease modified at or within residues 26-35 of a wild-type FMDV 3Cprotease where residues 26-35 of the wild-type protease comprise theamino acid sequence KTVA(I/L)CCATF (SEQ ID NO: 158, position 28underlined). One, two, three or more residues within this section of thewild-type FMDV 3C protease may be modified by substitution, deletion orinsertion. One example of such a variant is the V28K mutant of the FMDV3C protease. Polynucleotides encoding a variant containing the V28Ksubstitution include those selected from the group of polynucleotidesequences described by SEQ ID NO: 41, 43, 45, 47, 49, 51, 53, 55, 57,and 59 or selected from the group of polynucleotides encoding the aminoacid sequences of SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60.

A polynucleotide according to the invention may encode a FMDV 3Cprotease modified at or within residues 125-134 of a wild-type FMDV 3Cprotease where residues 125-134 of the wild-type protease comprise theamino acid sequence GRLIFSG(D/E)AL (SEQ ID NO: 156, position 127underlined). One, two, three or more residues within this section of thewild-type FMDV 3C protease may be modified by substitution, deletion orinsertion. One example of such a variant is the L127P mutant of the FMDV3C protease. Polynucleotides encoding a variant containing the L127Psubstitution include those selected from the group of polynucleotidesequences described by SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, 37,and 39 or selected from the group of polynucleotides encoding the aminoacid sequences of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

A polynucleotide according to the invention may encode a FMDV 3Cprotease modified at or within residues 138-150 of a wild-type FMDV 3Cprotease where residues 138-150 of the wild-type protease comprise theamino acid sequence D(I/L)VVCMDGDTMPF (SEQ ID NO: 157, positions 141 and142 underlined). One, two, three or more residues within this section ofthe wild-type FMDV 3C protease may be modified by substitution, deletionor insertion. Examples of such variants are the V141T and C142T mutantsof the FMDV 3C protease. Polynucleotides encoding a mutant containingthe V141T substitution include those selected from the group ofpolynucleotide sequences described by SEQ ID NO: 61, 63, 65, 67, 69, 71,73, 75, 77, and 79 or selected from the group of polynucleotidesencoding the amino acid sequences of SEQ ID NO: 62, 64, 66, 68, 70, 72,74, 76, 78 and 80; and polynucleotides encoding the C142T mutant (whichalso contain the L127P substitution) include those selected from thegroup of polynucleotide sequences described by SEQ ID NO: 81, 83, 85,87, 89, 91, 93, 95, 97, and 99 or selected from the group ofpolynucleotides encoding the amino acid sequences of SEQ ID NO: 82, 84,86, 88, 90, 92, 94, 96, 98 and 100.

A polynucleotide according to the invention may be a chimericpolynucleotide comprising a sequence encoding a modified or mutant FMDV3C protease and a sequence encoding a FMDV P1 protein or other proteincontaining cleavage sites recognized by the FMDV 3C protease, such as anon-naturally-occurring precursor polypeptide comprising FMDV viralproteins or other fused proteins separated by FMDV 3C protease cleavagesites. This is advantageous because a single polynucleotide moleculewill encode both the modified FMDV 3C protease and its substratefacilitating transformation or transfection of a host cell andcontrolling the relative numbers of copies of each coding sequence onthe single chimeric polynucleotide. A single chimeric polynucleotide canencode a modified FMDV 3C protease comprising one or more amino acidsubstitutions within residues 26-35, 125-134, or 138-150 of a wild-typeFMDV 3C protease, such as a mutant FMDV 3C protease comprising one ormore amino acid substitutions at positions 28, 127, 141 or 142 of anative 3C protease (e.g., L127P, V141T, and/or C142T), and furtherencode at least one FMDV P1 precursor polypeptide or other polypeptidesubstrate for the modified FMDV 3C protease.

Preferably, such a chimeric polynucleotide when transformed into andco-expressed in a host cell will (i) enhance transgene expression of theFMDV P1 precursor polypeptide (or other precursor substrate polypeptide)compared to an otherwise identical polynucleotide that encodes a FMDV 3Cprotease not comprising the one or more amino acid substitutions withinresidues 26-35, 125-134, or 138-150; (ii) increase an amount of FMDVVP0, VP1, VP2, VP3 and/or VP4 (or cleavage products of another substrateprecursor polypeptide) produced by the host cell compared to anotherwise identical polynucleotide that encodes a FMDV 3C protease notcomprising said the one or more amino acid substitutions within residues26-35, 125-134, or 138-150; (iii) increase the amount of eIF4A1translation factor in the host cell or decrease the amount ofproteolytically-cleaved eIF4A1 in the host cell compared to an otherwiseidentical polynucleotide that encodes a FMDV 3C protease not comprisingsaid the one or more amino acid substitutions within residues 26-35,125-134, or 138-150; or (iv) increase the amount of histone H3, nucleartranscription factor kappa B essential modulator (NEMO), orSrc-associated substrate in mitosis of 68 kDa (SAM68) in the host cellor decrease the amount of proteolytically-cleaved histone H3, nucleartranscription factor kappa B essential modulator (NEMO), orSrc-associated substrate in mitosis of 68 kDa (SAM68) in the host cellcompared to an otherwise identical polynucleotide that encodes a FMDV 3Cprotease not comprising said the one or more amino acid substitutionswithin residues 26-35, 125-134, or 138-150. Such polynucleotides may bedesigned, for example to adopt preferred codon usage of a particularhost cell, and inserted into vectors or polynucleotide constructssuitable for expression in a particular host cell or expression system,including eukaryotic and prokaryotic host cells.

Another associated embodiment to the polynucleotide of the invention asdescribed above is a vector or other polynucleotide construct, includingplasmids, minicircle vectors and viral or phage vectors, transposons, aswell as non-replicating (e.g. that do not contain an origin ofreplication), but transformable, polynucleotide constructs comprising apolynucleotide sequence according to the invention, which encode amodified FMDV 3C protease, or both a modified FMDV 3C protease and aFMDV P1 precursor protein or other engineered precursor proteincomprising cleavage sites recognized by the modified FMDV 3C protease.

Such a vector will generally contain at least one polynucleotidesequence encoding a mutant or variant FMDV 3C protease. Preferably thevector (or a combination of two or more vectors) will further compriseat least one sequence encoding FMDV P1 precursor protein or othersubstrate precursor protein of interest comprising FMDV 3C recognitionsites. Vectors comprising multiples of the FMDV 3C protease sequences ormultiples of FMDV P1 protein sequences (or other precursor proteinsequences) are also contemplated, such as vectors containing 2, 3, 4 ormore of such sequences. In another embodiment, the polynucleotideencoding a modified FMDV 3C protease and the polynucleotide encoding theFMDV P1 precursor protein or other precursor protein recognized by theprotease, may be placed on separate vectors suitable forco-transformation or co-transfection into a host cell.

The vector or polynucleotide construct of the invention may contain atleast one polynucleotide sequence encoding a FMDV P1 precursorpolypeptide from a FMDV O serotype, A serotype, C serotype, Asia 1serotype, SAT1 serotype, SAT2 serotype or SAT3 serotype; at least onepolynucleotide sequence encoding a FMDV P1 precursor polypeptide wherethe polynucleotide sequence is described by SEQ ID NO: 101, 103, 105,107, 109, 111, 113, 115 or 117; or at least one polynucleotide sequenceencoding a FMDV P1 precursor polypeptide that is at least 70, 75, 80,85, 90, 95, 96, 97, 98, or 99% similar or identical to SEQ ID NO: 102,104, 106, 108, 110, 112, 114, 116 or 118.

The vector or polynucleotide construct of the invention may furthercomprise at least one polynucleotide sequence encoding one or more ofFMDV viral proteins VP0, VP3, VP1; or VP1, VP2, VP3 and/or VP4 in thesame order that they are arranged in the FMDV P1 precursor protein or ina different order separated by cleavage sites recognized by the modifiedFMDV 3C protease.

The vector or polynucleotide construct of the invention may furthercomprise at least one promoter or other transcription regulatoryelement, at least one prokaryotic or eukaryotic translation initiationsequence or other translation regulatory element, at least onetranslational interrupter sequence, at least one reporter gene, or atleast one selectable marker, such as an antibiotic resistance gene,operatively linked to the polynucleotide sequence encoding the modifiedFMDV 3C protease or other precursor protein of interest encoded thevector or polynucleotide construct. Other elements, such as atranslation interrupter sequence such as 2A or Δ1D2A sequence may beincorporated into a vector or polynucleotide construct at places whereprotein cleavage is desired, such as before or after a tag, marker orindicator protein such as GLuc and/or SGLuc.

A vector or polynucleotide construct may be selected based on itscapacity to be expressed by a particular host cell or expression system,such as in a eukaryotic or prokaryotic cell. Examples of such vectorsinclude those that can be transformed into and expressed bySaccharomyces cerevisiae, Pichia pastoris, or another yeast or funguscell; Arabidopsis thaliana, Chlamydomonas reinhardtii, by Glycine max,Nicotiana benthamiana, Nicotiana tabacum, Oryza sativa, Zea mays oranother plant cell; by Spodoptera frugiperda, Drosophila melanogaster,Sf9, Sf21, or another insect cell; by a vertebrate cell; by HEK-293-T(human kidney embryo) cell, LF-BK (porcine cell), LF-BK αV/β6 cell, orby another mammalian cell; by a host cell derived from a mammal or otheranimal susceptible to FMDV infection. Alternatively, the vector,including phage vectors, may be selected to transform or transfect intoand be expressed by a prokaryotic cell including Bacillus, Lactococcus,Streptomyces, Rhodococcus, Corynebacterium, Mycobacterium or in anothergram-positive prokaryote; or Escherichia, Pseudomonas or anothergram-negative prokaryote.

A vector or construct may constitute a minicircle vector, a replicationdeficient adenovirus vector, a vaccinia virus vector, or other viralvector that expresses the modified FMDV 3C protease and, optionally, aFMDV P1 precursor polypeptide or other precursor protein of interest ina host cell. Such host cells include those directly obtained from anorganism, including immunocytes, such as lymphocytes or macrophages,stem cells or muscle cells, as well as cell lines passaged in vitroderived from such cells.

Another aspect of the invention compatible with the polynucleotides andvectors/polynucleotide constructs of the invention as disclosed above isa host cell that can be transformed or transfected with a polynucleotideor vector according to the invention and used to express the modifiedFMDV 3C protease and other polypeptides of interest, such as FMDV P1precursor protein. Such a host cell may be transformed or transfected toexpress only the modified FMDV 3C protease or may be co-transformed orco-transfected with vectors or polynucleotide constructs expressing boththe modified 3C protease the FMDV P1 precursor protein or othercleavable protein of interest.

The host cell of the invention may contain a vector or polynucleotideconstruct further comprising at least one promoter or othertranscription regulatory element, at least one prokaryotic or eukaryotictranslation initiation sequence or other translation regulatory element,at least one translational interrupter sequence, at least one reportergene, or at least one selectable marker, such as an antibioticresistance gene, operatively linked to the polynucleotide sequenceencoding the modified FMDV 3C protease or another protein of interestencoded by polynucleotides in said vector.

The host cell of the invention may be transformed or transfected with asingle vector or polynucleotide construct or with multiple vectors ormultiple polynucleotide constructs, encoding the modified FMDV 3Cprotease and FMDV P1 precursor polypeptide or other cleavablepolypeptide of interest that contains cleavage sites recognized by themodified FMDV 3C protease. The host cell may express a recombinant afusion protein or chimeric protein comprising a FMDV P1 precursorpolypeptide or one or more, or a set of FMDV viral protein(s) such asVP0, VP3, and VP1, or VP1, VP2, VP3 and VP4, a translation interruptersequence such as 2A or Δ1D2A, a linker or spacer peptide, a His-tag orFLAG-tag or other protein tag, a reporter protein, or a luminescentsequence such as GLuc and/or SGLuc. A host cell according to theinvention may express FMDV proteins from various serotypes, such as aFMDV P1 precursor polypeptide from a FMDV O serotype, A serotype, Cserotype, Asia 1 serotype, SAT1 serotype, SAT2 serotype or SAT3 serotypeor a FMDV P1 precursor polypeptide described by SEQ ID NO: 102, 104,106, 108, 110, 112, 114, 116 or 118, or a FMDV P1 protease at least 70,75, 80, 85, 90, 95, 96, 97, 98, or 99% similar or identical thereto.

The host cell of the invention may be obtained from or derived from aeukaryotic cell. Examples of such host cells include Saccharomycescerevisiae, Pichia pastoris, or another yeast or fungus cell;Arabidopsis thaliana, Chlamydomonas reinhardtii, Glycine max, Nicotianabenthamiana, Nicotiana tabacum, Oryza sativa, Zea mays or another plantcell; Spodoptera frugiperda, Drosophila melanogaster, Sf9, Sf21, oranother insect cell; a vertebrate cell; HEK-293-T (human kidney embryo)cell, LF-BK (porcine cell), LF-BK αV/β6 cell, or another mammalian cell;by a host cell derived from a mammal or other animal susceptible to FMDVinfection.

Alternatively, a host cell may be a prokaryotic cell such as Bacillus,Lactococcus, Streptomyces, Rhodococcus, Corynebacterium, Mycobacteriumor another gram-positive prokaryote; or Escherichia, Pseudomonas oranother gram-negative prokaryote.

The host cell may be one suitable for transformation by the vectors andpolynucleotide constructs disclosed herein or by a minicircle vector, areplication deficient adenovirus vector, a vaccinia virus vector, orother viral vector that expresses the modified FMDV 3C protease and,optionally, a FMDV P1 precursor polypeptide or other protein of interestbesides the modified FMDV 3C protease in a host cell. Such host cellsinclude those directly obtain from an organism, including stem cells,epithelial cells, or muscle cells, as well as cells passaged in vitroderived from such cells.

A host cell of the invention may contain a vector or polynucleotideconstruct that encodes the recombinant FMDV P1 precursor polypeptidewhich will be expressed by the host cell and cleaved by the recombinantFMDV 3C protease also expressed by the host cell. It may contain avector or construct encoding a modified foot-and-mouth disease (FMDV) 3Cprotease as disclosed herein which comprises one or more amino acidsubstitutions within residues 26-35, 125-134, or 138-150 of a FMDV 3Cprotease, and at least one polynucleotide sequence that encodes an FMDVP1 precursor polypeptide, wherein said host cell expresses a higheramount of FMDV P1 precursor polypeptide than an otherwise identical hostcell containing a vector or polynucleotide construct that encodes a FMDV3C protease not comprising said the one or more amino acid substitutionswithin residues 26-35, 125-134, or 138-150; or a host cell thatexpresses more VP0, VP3, or VP1 (or VP2, VP4, VP3 and VP1) than anotherwise identical host cell containing a vector or polynucleotideconstruct that encodes a FMDV 3C protease not comprising the one or moreamino acid substitutions within residues 26-35, 125-134, or 138-150; ora host cell that produces more FMDV VLPs than an otherwise identicalhost cell containing a vector or polynucleotide construct that encodes aFMDV 3C protease not comprising said the one or more amino acidsubstitutions within residues 26-35, 125-134, or 138-150. For example, ahost cell of the invention may produce 1.05, 1.1, 1.25, 1.5, 1.75, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20 or more times FMDV P1 precursor protein,VP0, VP1, VP2, VP3, VP4, or VLPs that an otherwise identical host cellexpressing a corresponding not mutated FMDV 3C protease.

A host cell according to the invention may contain a vector orpolynucleotide construct encoding a modified foot-and-mouth disease(FMDV) 3C protease comprising one or more amino acid substitutionswithin residues 26-35, 125-134, or 138-150, and at least onepolynucleotide sequence that encodes an FMDV P1 precursor polypeptide,wherein said host cell expresses or contains (i) a higher amount ofeIF4A1 translation factor, histone H3, nuclear transcription factorkappa B essential modulator (NEMO), or Src-associated substrate inmitosis of 68 kDa (SAM68) or (ii) produces or contains a decreasedamount of proteolysed eIF4A1, histone H3, nuclear transcription factorkappa B essential modulator (NEMO), or Src-associated substrate inmitosis of 68 kDa (SAM68) compared to the amounts expressed or presentin an otherwise identical host cell containing a vector orpolynucleotide construct that encodes a FMDV 3C protease not comprisingthe one or more amino acid substitutions within residues 26-35, 125-134,or 138-150.

A host cell according to the invention may also contain a vector orpolynucleotide construct encoding a modified foot-and-mouth disease(FMDV) 3C protease comprising one or more amino acid substitutionswithin residues 26-35, 125-134, or 138-150, and at least one othervector or polynucleotide construct encoding an FMDV P1 precursorpolypeptide or other polypeptide of interest besides the FMDV 3Cprotease. Host cells which express a modified foot-and-mouth disease(FMDV) 3C protease and/or an FMDV P1 precursor polypeptide or otherpolypeptide of interest from their chromosomal DNA are alsocontemplated. For example, a host cell may express a modified FMDV 3Cprotease from its chromosomal DNA and express a FMDV P1 precursor fromvector DNA, or vice versa.

Another aspect of the invention constitutes a method for expressingand/or processing FMDV P1 precursor polypeptide (or other precursorpolypeptide of interest) into FMDV viral proteins (or smaller proteinsof interest) comprising culturing a host cell according to the inventionin a suitable medium and recovering viral proteins VP0, VP1, VP2, VP3 orVP4 or FMDV virus-like particles or other cleavage products of a proteinor polypeptide of interest; wherein said host cell expresses a modifiedFMDV 3C protease, which contains one or more amino acid substitutionswithin residues 26-35, 125-134 or 138-150 of a native FMDV 3C protease,and said host cell also expresses an FMDV P1 precursor polypeptide. Sucha method may be employed to produce one or more or a set of FMDV viralproteins or VLPs. For example, it may be used to produce and recoverVP0, VP1 and VP3 or VP1, VP2, VP3 and VP4. Such a method may result inthe cleavage by the modified 3C protease of at least 10, 20, 30, 40, 50,60, 70, 80, 90 or 100% of the FMDV P1 precursor polypeptide or otherprecursor polypeptide of interest expressed by a host cell according tothe invention.

In another embodiment, the method according to the invention may cleaveless of one or more host cell proteins which are cleaved by acorresponding unmodified FMDV 3C protease which does not contain one ormore amino acid substitutions within residues 26-35, 125-134 or 138-150of a corresponding unmodified FMDV 3C protease amino acid sequence. Forexample, the method according to the invention using a modified 3Cprotease may cleave less than 10, 20, 30, 40 or 50% of one or more hostcell proteins than an otherwise identical method using the correspondingunmodified FMDV 3C protease. Such host cell proteins include, but arenot limited to, eIF4A1, histone H3, nuclear transcription factor kappa Bessential modulator (NEMO), or Src-associated substrate in mitosis of 68kDa (SAM68). Thus, host cells employed in a method according to theinvention, which express a modified FMDV 3C protease, may contain (i) anincreased amount of not proteolyzed eIF4A1, histone H3, nucleartranscription factor kappa B essential modulator (NEMO), orSrc-associated substrate in mitosis of 68 kDa (SAM68) or (ii) adecreased amount of proteolysed eIF4A1, histone H3, nucleartranscription factor kappa B essential modulator (NEMO), orSrc-associated substrate in mitosis of 68 kDa (SAM68) compared to hostcells used in an otherwise identical method which uses host cellsexpressing an unmodified FMDV 3C protease, such as a 3C protease whichdoes not contain one or more amino acid substitutions within residues26-35, 125-134 or 138-150.

A method according to the invention may produce and/or allow recovery ofmore FMDV P1 precursor polypeptide, more of at least one kind of FMDVviral protein, such as VP0, VP1, VP2, VP3, or VP4, or more VLPs (or morenon-P1 precursor polypeptides and their proteolytic or quaternaryproducts) than an otherwise identical method using a host cellcontaining a corresponding unmodified FMDV 3C protease which does notcontain one or more amino acid substitutions within residues 26-35,125-134 or 138-150 of a wild-type FMDV 3C protease.

In some embodiments, a method according to the invention will comprisingculturing a host cell containing a vector or polynucleotide constructcontaining a translation interrupter sequence, such as a 2A sequence,and optionally a protein that can be secreted from the cell, such asGLuc or SGLuc. A translation interrupter sequence permits interruptionof protein translation and effective cleavage at a site not necessarilyrecognized by a 3C protease such as a site between a FMDV P1 protein anda reporter protein or luminescent protein, such as GLuc, SGLuc or otherluciferase protein which, optionally, can be secreted. Interruption oftranslation by 2A produces a polypeptide not having an N-terminal Metresidue.

This aspect of the invention includes embodiments where the vector orpolynucleotide construct in the host cell encodes a modified 3Cprotease, a 2A or other translation interrupter sequence, and an FMDV P1precursor polypeptide, and optionally at least one of GLuc, SGLuc orother luciferase which can be secreted; and embodiments where the hostcell comprises a vector or polynucleotide construct expressing a fusionprotein comprising a modified 3C protease and an FMDV P1 precursorpolypeptide and at least one of GLuc, SGLuc or other luciferase whichcan be secreted, and optionally at least one of a 2A, Δ1D2A, or othertranslation interrupter sequence.

In these embodiments a 2A sequence will generally be arranged betweensegments of a fusion protein to be separated and produce a downstream(toward the C-terminal of the fusion protein) protein or proteinfragment without an N-terminal Met residue. The N-terminal andC-terminal portions of such a fusion protein may contain a FMDV 3Cprotease segment, FMDV P1 precursor protein (or other precursor proteinof interest) segment, and a GLuc or SGLuc segment in any order,including where the GLuc or SGLuc segments appear at the C-terminal orN-terminal of the fusion protein and are separated from the remainder ofthe fusion protein by a 2A-type translation interrupter site. Separationof a protein that can be secreted by the cell, such as GLuc or SGLuc,from a longer fusion protein molecule that also encodes the FMDV P1precursor or other precursor polypeptide of interest, and/or themodified FMDV 3C protease, provides a convenient means to monitorrecombinant protein expression by measuring levels of the secretedprotein in an extracellular medium.

Another aspect of the invention constitutes a modified foot-and-mouthdisease (FMDV) 3C protease comprising one or more amino acidsubstitutions within residues 26-35, 125-134 or 138-150 of a wild-typeFMDV 3C protease. A modified foot-and-mouth disease (FMDV) 3C proteaseaccording to the invention may be, but is not limited to one, that is atleast 70, 80, 90, 95, 96, 97, 98, 99% identical or similar to at leastone wild-type protease from FMDV O serotype, A serotype, C serotype,Asia 1 serotype, SAT1 serotype, SAT2 serotype, or SAT3 serotype, to a 3Cprotease encoded by a polynucleotide sequence described by SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, and 19, or to an amino acid sequencedescribed by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. Such amodified protease may contain all or some of the cysteine or prolineresidues found in a native FMDV protease, such as cysteine residuesforming disulfide bridges in a native FMDV 3C protease. In someembodiments it will contain cysteine residues at positions 51 and 163,residues H46, D84 and C163, or will not contain glutamic acid (E) atposition 126 (R126E) or will not contain serine at position 133 (A133S),or have combinations of these retained or substituted residues.

In most embodiments, the modified FMDV 3C protease variant, orfunctionally active portions of such a protease will exhibit proteolyticactivity on a FMDV P1 precursor polypeptide or on other polypeptidescontaining FMDV 3C protease recognition sites. A modified FMDV 3Cprotease is preferably less toxic to, less disruptive to proteinexpression, less growth inhibitory to, or exhibits attenuatedproteolytic activity against host cell proteins compared to acorresponding unmodified FMDV 3C protease. However, in some embodiments,the variant FMDV 3C protease can have attenuated or no proteolyticactivity against FMDV P1 protein, but exhibit other useful properties,such as immunogenicity, antigenicity or the ability to modulate cleavageor processing of FMDV P1 or other proteins recognized by native or otherfunctionally active FMDV 3C proteases.

A FMDV 3C protease according to the invention may be modified at orwithin residues 26-35 of a wild-type FMDV 3C protease where residues26-35 of the wild-type protease comprise the amino acid sequenceKTVA(I/L)CCATF (SEQ ID NO: 158, position 28 underlined). One, two, threeor more residues within this section of the wild-type FMDV 3C proteasemay be modified by substitution, deletion or insertion. One example ofsuch a variant is the V28K mutant of the FMDV 3C protease. Variantscontaining the V28K substitution include those encoded by thepolynucleotide sequences described by SEQ ID NO: 41, 43, 45, 47, 49, 51,53, 55, 57, and 59 or those comprising the amino acid sequences of SEQID NO: 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60.

A FMDV 3C protease according to the invention may be modified at orwithin residues 125-134 of a wild-type FMDV 3C protease where residues125-134 of the wild-type protease comprise the amino acid sequenceGR{right arrow over (L)}IFSG(D/E)AL (SEQ ID NO: 156, position 127underlined). One, two, three or more residues within this section of thewild-type FMDV 3C protease may be modified by substitution, deletion orinsertion. One example of such a variant is the L127P mutant of the FMDV3C protease. Variants containing the L127P substitution include thoseencoded by the polynucleotide sequences described by SEQ ID NO: 21, 23,25, 27, 29, 31, 33, 35, 37, and 39 or those comprising the amino acidsequences of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

A FMDV 3C protease according to the invention may be modified at orwithin residues 138-150 of a wild-type FMDV 3C protease where residues138-150 of the wild-type protease comprise the amino acid sequenceD(I/L)VVCMDGDTMPF (SEQ ID NO: 157, positions 141 and 142 underlined).One, two, three or more residues within this section of the wild-typeFMDV 3C protease may be modified by substitution, deletion or insertion.Examples of such variants are the V141T and C142T mutants of the FMDV 3Cprotease.

A variant containing the V141T substitution may be encoded by apolynucleotide sequence described by SEQ ID NO: 61, 63, 65, 67, 69, 71,73, 75, 77, and 79 or comprise an amino acid sequence of SEQ ID NO: 62,64, 66, 68, 70, 72, 74, 76, 78 and 80.

A variant containing the C142T substitution (which also contains theL127P substitution) may be encoded by a polynucleotide sequencedescribed by SEQ ID NO: 81, 83, 85, 87, 89, 91, 93, 95, 97, and 99 orcomprise an amino acid sequence of SEQ ID NO: 82, 84, 86, 88, 90, 92,94, 96, 98 and 100.

Another aspect of the invention is a composition comprising a modifiedFMDV 3C protease and a pharmaceutically acceptable excipient oradjuvant, or a buffer or solution suitable for performing proteolysisusing a modified FMDV 3C protease. Such a composition may be used toinduce or detect immune responses against the 3C protease, such ashumoral (e.g., antibody) or cellular immune responses (e.g., T-cellresponses) directed against the 3C protease. Such a composition mayfurther contain precursor polypeptides, such as FMDV P1 precursor, thatcontain sites recognized by the modified FMDV 3C protease and be in aform useful for processing precursor polypeptides in vitro.

Another aspect of the invention represents a FMDV P1 precursorpolypeptide, P1 polypeptide, VP0, VP1, VP2, VP3 or VP4 protein or FMDVvirus-like particle produced by a host cell or method described herein.Such a FMDV P1 precursor polypeptide, P1 polypeptide, VP0, VP1, VP2, VP3or VP4 protein or FMDV virus-like particle may be produced by aprokaryotic or a eukaryotic expression system and have featurescharacteristics of those expression systems, such as the absence ofglycosylation when expressed by a prokaryote, or the presence ofglycosylation or other post-translation modifications provided duringeukaryotic expression.

Another aspect of the invention is an antigen, immunogen or vaccinecomprising the FMDV P1 precursor polypeptide, P1 polypeptide, VP0, VP1,VP2, VP3 or VP4 protein or FMDV virus-like particles produced using themodified FMDV 3C protease disclosed herein in combination with asuitable carrier, excipient or adjuvant. Such products are useful fordetecting immune responses against FMDV, such as FMDV-specificantibodies or FMDV-specific T-cells. Such antigens, immunogens orvaccines also may be used to induce an immune response against FMDV,vaccinate a subject against FMDV, or reduce the severity of an FMDVinfection by administering them to a subject in need thereof such as asubject infected with or at risk of infection by FMDV. A FMDV P1precursor polypeptide, P1 polypeptide, VP0, VP1, VP2, VP3 or VP4 proteinor FMDV virus-like particle produced by a method according to theinvention may be administered to a subject in need thereof

Alternatively, a composition comprising a vector or polynucleotideconstruct encoding a modified FMDV 3C protease polypeptide and a P1precursor protein may be administered to a subject in need thereof, forexample, into muscle tissue of an animal susceptible to FMDV infection,where the P1 precursor polypeptide can be expressed and proteolyticallyprocessed in vivo by the co-expressed modified FMDV 3C protease.

Another aspect of the invention involves chimeric polynucleotidesencoding fusion proteins comprising FMDV segments and translationtermination interrupters, such as 2A-like sequences, and reporterproteins such as GLuc or SGLuc and the encoded fusion proteins per se.Such a polynucleotide may encode a fusion protein comprising at leastone of (i) a 2A, Δ1D2A, or other translation interrupter sequence, andat least one GLuc, SGLuc or other luciferase which can be secretedand/or at least one enzyme or other polypeptide of interest which can besecreted. Such a polynucleotide may encode at least one GLuc, SGLuc orother luciferase which can be secreted and/or at least one enzyme orother polypeptide of interest which can be secreted that does not havean N-terminal methionine residue by virtue of translation interruptionby 2A or a 2A-like sequence. Examples of such chimeric polynucleotideconstructs encoding fusion proteins include the polynucleotides havingsequences described by SEQ ID NO: 147, 148, 149, 150, 151, or 152 andvariant polynucleotides that have sequences that are at least 70, 80,90, 95 or 99% identical or similar to the sequences described by SEQ IDNO: 147, 148, 149, 150, 151, or 152 and which retain the functionalityof the FMDV, 2A or other translation interrupter, and GLuc, SGLuc orother reporter protein segments. Examples of fusion proteins includethose encoded by SEQ ID NO: 147, 148, 149, 150, 151, or 152 or theirpolynucleotide variants, which are described by SEQ ID NO: 213, 214,215, 216, 217 or 218 as well as variant amino acid sequences that are atleast 70, 80, 90, 95 or 99% identical or similar to a sequence of SEQ IDNO: 213, 214, 215, 216, 217 or 218 and which retain the functionality ofthe FMDV, 2A or other translation interrupter, and GLuc, SGLuc or otherreporter protein segments.

The polynucleotide according to this aspect of the invention may beincorporated into a vector or polynucleotide construct, such as thevectors or polynucleotide constructs described herein and such a vectoror polynucleotide construct may be transformed into, or transfectedinto, a suitable host cells such as those disclosed herein A relatedaspect of the invention is a method for producing an enzyme or otherpolypeptide of interest that can be secreted from the cell, comprisingculturing the host cell described above in suitable medium andrecovering the enzyme or other polypeptide of interest outside of thecell. The protein that can be secreted may optionally lack an N-terminalMet residue, for example, by action of a 2A-like translation interruptersequence.

Such a method may employ a host cell containing a vector orpolynucleotide construct that encodes a fusion protein comprising (i) aprotein of interest, (ii) at least one of a 2A, Δ1D2A, or othertranslation interrupter sequence, and/or (iii) at least one polypeptidethat can be secreted from the cell selected from the group consisting ofGLuc, SGLuc or other luciferase, at least one enzyme that can besecreted from the cell, and at least one other detectable polypeptidewhich can be secreted from the cell, and may further comprise monitoringexpression of the protein of interest by measuring the amount of the atleast one polypeptide that can be secreted from the cell outside thehost cell. The secreted protein may lack an N-terminal Met residuedepending on the relative location of a 2A interrupter sequence.

The protein of interest may be a modified FMDV 3C protease, a FMDV P1precursor polypeptide, or some other polypeptide of interest and may beincorporated as an antigen, immunogen or vaccinogen into a compositionfurther comprising at least one pharmaceutically acceptable carrier,adjuvant, or excipient. Such a composition may be used for immunizing asubject by administering it to a subject in need thereof. For example,when the protein of interest is an FMDV P1 polypeptide or FMDV viralprotein or VLP, it may be compounded into a composition with suitableexcipients or adjuvants and administered to a subject susceptible toFMDV infection.

Proteins of interest produced by this method may be used for detectingan antibody, immunocyte (e.g. lymphocyte, macrophage) or other agentthat binds to or interacts with the enzyme or other polypeptide ofinterest that can be secreted by the cell by contacting a samplecontaining said antibody, immunocyte or other agent with the enzyme orother polypeptide of interest, which may be expressed so as to lack anN-terminal Met residue, and detecting or quantifying complex formationthereby detecting said antibody, immunocyte or other agent.

Another aspect of the invention is a method for producing a polypeptideof interest that does not have an N-terminal methionine residuecomprising transforming a host cell with a vector encoding a 2A, Δ1D2A,or other translation interrupter sequence operably linked to apolynucleotide encoding the polypeptide of interest and recovering thepolypeptide of interest without the N-terminal methionine residue. Theprotein of interest may be recovered from the cell or, when it iscapable of being secreted, from an extracellular medium. The vector insaid method may be one that further encodes at least one of GLuc, SGLucor other luciferase, enzyme or polypeptide of interest that can besecreted by the cell.

Another aspect of the invention constitutes a method for producing anenzyme or other polypeptide of interest that can be secreted by the cellcomprising transforming a host cell with a vector encoding said enzymeor other polypeptide of interest fused to at least one of a 2A, Δ1D2A,or other translation interrupter sequence. The enzyme or polypeptide ofinterest that can be secreted by the cell in said method may be selectedto be GLuc or SGLuc.

Reference through the specification to “one embodiment” or “anembodiment” means that a particular feature, structure, orcharacteristic described in connection with an embodiment is included inat least one embodiment of the subject matter disclosed. Thus, theappearance of the phrases “in one embodiment” or “in an embodiment” invarious places throughout the specification is not necessarily referringto the same embodiment. Further, the particular features, structures orcharacteristics may be combined in any suitable manner in one or moreembodiments.

EXAMPLES

The examples below are intended to further illustrate various protocols,including protocols for preparing and characterizing mutant FMDV 3Cproteases, transgene expression cassettes carrying the mutant FMDV 3Cproteases, vectors carrying the transgene expression cassettes, hostcells transformed with the vectors, virus-like particles assembled andformed inside the host cells, DNA constructs or chimeras fusing aGaussia luciferase gene (GLuc) or a super-luminescent Gaussia luciferase(SGLuc) with a foot-and-mouth disease virus (FMDV) translationalinterrupter sequence and an FMDV virus-like particle or VLP-expressingconstruct, and a mechanism for enhancement for transgene output bymutant FMDV 3C proteases. These examples are not intended to limit thescope of the claims. While these examples are provided for explanatorypurposes, these should not be considered the only examples. Additionalexamples will be apparent based on the teachings of the presentdisclosure.

Overview of Examples 1-15

In the following Examples 1-15, in order to examine the effect of theexpression of wild-type and mutant FMDV 3C proteases on transgeneoutput, a luminescence assay that utilizes a chimera (Gluc-3C) ofGaussia princeps luciferase (GLuc) and an FMDV 3C protease (3C) wasdeveloped to monitor and quantify the cytotoxic effect that FMDV 3Cprotease expression has on transfected mammalian cells and totaltransgene output of transfected cells expressing the FMDV 3C protease.GLuc (SEQ ID NO: 201) is a small, naturally secreted luciferase of 185amino acids (encoded by SEQ ID NO: 145 or 200). GLuc has a higherintensity when compared to firefly or Renilla luciferases making itideal for studies which may involve only a small amount of peptide. Amutation of amino acids 89 and 90 in GLuc produces a super luminescentGLuc variant known as SGLuc (SEQ ID NO: 203), encoded by SEQ ID NO: 146or 202 useful for examination of low levels of protein expression. GLucis stable at 37° C. for extended periods of time which allows for abuildup of active GLuc in cell culture media further enhancing detectionthresholds. These properties make SGLuc suitable for examination ofprotein expression over time when using transfected cell cultures.

The GLuc-3C constructs in Examples 1-15 utilize a Δ1D2A translationalinterrupter sequence (SEQ ID NO: 119) derived from the FMDV 2A proteinto allow for individual expression of both GLuc and 3C. The 2Atranslational interrupter sequence results in equimolar quantities ofeach peptide being produced from a single open reading frame. Efficiencyof 2A mediated cleavage depends upon the sequence present with the mostefficient cleavage sequences incorporating portions of the C-terminus ofthe FMDV 1D protein. Inclusion of an 11 amino acid portion of theC-terminus of 1D (Δ1D2A) to the N-terminus of 2A and a proline residueto the C-terminus of 2A, as shown in FIG. 4A, makes 2A mediatedtranslational interruption efficiency insensitive to the sequenceupstream allowing for any protein to be present without detriment to 2Aactivity. The usage of the Δ1D2A sequence in chimeric constructs allowsthe total transgene output in response to FMDV 3C protease expression tobe monitored, by measuring for the presence of luciferase activity incell culture media, as shown in FIG. 4B.

Examples 1-15 show that the five different FMDV 3C protease singlemutations (V28K, L127P, V141T, C142T and C163A) have diverse effects ontotal transgene output. The effect on transgene output was consistentregardless of whether or not the FMDV P1 polypeptide precursor waspresent in the construct. The sole exception was the inversion of theC163A and L127P samples in all luciferase assays conducted to monitortotal transgene output. This inversion suggests that the presence of alarge amount of unprocessed P1 polypeptide precursor may have adetrimental effect on transgene output. It should be noted however thatany detrimental effect that the unprocessed P1 may have is minor whencompared to the detrimental effect of expressing the wild-type FMDV 3Cprotease.

The C163A mutation is a knock-out mutation that removes all proteolyticactivity of the FMDV 3C protease. The C142T mutation has been previouslyused in conjunction with an HIV-ribosomal frameshift sequence to downregulate overall 3C expression.

All mutations except C163A showed varying capabilities of processing ofthe P1 polypeptide precursor. With L127P, which is a mutation/amino acidsubstitution to the B₂ β-strand and is a surface residue having noproximity to the substrate binding cleft and the active site of theprotease, the present inventors have surprisingly found that the mutantFMDV 3C protease is retaining the ability to fully process the P1polypeptide while dramatically enhancing transgene output (i.e. nottoxic to the host cells).

The combination of the L127P and C142T mutations produced a constructthat was able to further enhance transgene output from the L127P andC142T single mutants and approximately 25× higher than the wild-type.The L127P/C142T double mutant produced an abundance of VLP crystallinearrays when HEK293-T transfected with the double mutant construct wasexamined with transmission electron microscopy (TEM).

Example 1: Insertion of GLuc into pTarget

Template DNA for GLuc was PCR amplified using OneTaq 2× Master Mix withStandard Buffer (New England Biolabs) and primers AscI-Kzk-Gluc-F (SEQID NO: 182) and Gluc-NS-NheI-R (SEQ ID NO: 181) per manufacturer'sinstructions. Insertion into the pTarget vector (Promega) followedmanufacturer's instructions for T/A cloning. Transformants were platedon LB Agar plates with 100 ug/ml carbenicillin (Teknova). To confirmmutation-free insertion, the plasmids were sequenced using primers T7(SEQ ID NO: 179) and Seq-R (SEQ ID NO: 180). Sequencing data wasanalyzed using the Sequencher 4.8 program (Genecodes).

Example 2: Construction of pTarget-GLuc-Δ1D2A

The Δ1D2A translational interrupter sequence used was derived from theFMDV A24 serotype sequence and flanked by NheI and XmaI restrictionsites. Restriction digestions were performed on both the pTarget-GLucvector and the Δ1D2A translational interrupter sequence using XmaI andNheI-HF restriction enzymes (New England Biolabs) per manufacturer'sinstructions. Both vector and insert were purified using a PCRpurification kit (Qiagen). Ligation of the Δ1D2A sequence into the cutpTarget-GLuc vector was performed using T4 DNA ligase (Roche) as permanufacturer's instructions. Ligation reaction mix was cloned into NEB5-alpha Competent E. coli (New England Biolabs) as per manufacturer'sinstructions. Sequencing was performed to confirm insertion as describedpreviously in Example 1.

Example 3: Construction of pTarget-Gluc-Δ1D2A-3C

Amplification of 3C nucleic acids from an FMDV Asia Lebanon 89(Accession # AY593798, SEQ ID NO: 1) non-infectious template wasperformed using OneTaq 2× Master Mix with Standard Buffer (New EnglandBiolabs) as per manufacturer's instructions and using primers XmaI-3C-F(SEQ ID NO: 183) and 3C-NotI-R (SEQ ID NO: 174). PCR product waspurified using a PCR purification kit (Qiagen). Both the 3C PCR productand pTarget Gluc-Δ1D2A vector were digested with XmaI and NotI-HFrestriction enzymes (New England Biolabs) as per manufacturer'sinstructions. Ligation and cloning were performed as described inExample 2. Sequencing was performed as described in Example 1.

Example 4: Production of Mutant Nucleotide Sequences Encoding MutantFMDV 3C Proteases

The L127P Asia Lebanon 89 mutant nucleotide sequence (SEQ ID NO: 21) wascreated through random error during PCR amplification of 3C andidentified during the sequencing process.

The V28K Asia Lebanon 89 (SEQ ID NO: 41), V141T Asia Lebanon 89 (SEQ IDNO: 61), and C163A Asia Lebanon 89 (SEQ ID NO: 209) mutant nucleotidesequences were created by using the GENEART® Site-Directed MutagenesisSystem (Invitrogen) with the following primer sets:

3CLeb89 V28K-MF (SEQ ID NO: 163) and 3CLeb89 V28K-MR (SEQ ID NO: 164),

3CLeb89 V141T-MF (SEQ ID NO: 165) and 3CLeb89 V141T-MR (SEQ ID NO: 166),

3C C142T-MF (SEQ ID NO: 167) and 3C C142T-MR (SEQ ID NO: 168), or

3C C163A-MF (SEQ ID NO: 169) and 3C C163A-MR (SEQ ID NO: 170) forrespective mutations.

The L127P Asia Lebanon 89 mutant nucleotide sequence (SEQ ID NO: 21)could also be alternatively constructed using primers 3C L127P-MF (SEQID NO: 171) and 3C L127P-MR (SEQ ID NO: 172) with the GENEART®Site-Directed Mutagenesis System.

Creation of the L127P/C142T Asia Lebanon 89 double mutant nucleotidesequence (SEQ ID NO: 81) was performed using the GENEART® Site-DirectedMutagenesis System on the previously constructed mpTargetO1P1-3C(C142T)-SGLuc construct as a template and using the 3C L127P-MF(SEQ ID NO: 171) and 3C L127P-MR (SEQ ID NO:172) primers.

The locations of the FMDV 3C protease residues subjected to mutation andsubstitution, namely V28, L127, V141, C142 and C163 are indicated in theribbon diagram of the protein of FIG. 5.

Site-directed mutagenesis PCR reactions were performed according tomanufacturer's instructions with optional slight modifications asrecognized as appropriate by a person of ordinary skill in the art. Eachsite-directed mutagenesis PCR reaction contains 45 μl of Accuprime PfuSupermix (Life Technologies), 5 μl of 10× enhancer (Invitrogen), 1 μl ofDNA methylase (Invitrogen), 0.25 μl of 200×S-adenosine methionine SAM(Invitrogen), 0.1 μl of pTarget Gluc-Δ1D2A-3C and 250 ng of each primer.The mutagenesis PCR reactions were carried out using the followingparameters: 37° C. for 20 min; 94° C. for 2 min; then 35-40 cycles of(94° C. for 20 s, 57° C. for 30 s, 68° C. for 3 min and 30 s); and 68°C. 5 min. The recombination reaction and E. coli transformations wereperformed as suggested by the manufacturer. Sequencing was performed aspreviously described.

Example 5: Transfection

HEK293-T cells (passage 41) were grown on Costar 6 Well Clear TC-TreatedMultiple well plates (Corning Incorporated) in 293 growth media (1×minimum essential media or MEM media, 10% fetal bovine serum, 1% 100×GLutaMax™ media supplement, 1% MEM-NEAA (Non-essential amino acids), and1% Antibiotic-antimycotic solution). Cells were rinsed with 2 ml of 1×Dulbecco's phosphate-buffer saline or DPBS (Gibco) and 2 mL of freshmedia added to each well at roughly 80% confluence. Transfections wereperformed using 4 μg of plasmid DNA (pTarget Gluc-Δ1D2A-3C construct)and 10 μl of Lipofectamine 2000 per manufacturer's instructions.Transfected cells were placed in a 37° C. CO₂ incubator for 24 h.

Example 6: Luciferase Assay

After the incubation media was removed from each well containing atransfection reaction, the luciferase activity of each well was measuredusing a 96-well BioSystems Veritas luminometer (Turner Biosystems) with20 μl of sample in each well. Readings were taken immediately uponinjection of 25 μl of 50 μg/μl coelenterazine solution (NanoLightTechnologies, Pinetop Ariz.) using an integration time of 0.5 s bothbefore and after injection of substrate. Pre-injection readings wereused to establish a baseline of light emission at the time of injectionand subtracted from the post-injection values for data analysis.Replicates were averaged together to give the overall luciferase readingin relative light units per half second (RLU/0.5 s). A total of sevenreplicates were used for each sample.

Example 7: Construction of O1P1-3C-SGLuc Expression Constructs

The O1P1-3C-SGLuc constructs were prepared using a modified pTarget(mpTarget) vector. Modifications included decreasing overall vector sizeas well as removal of the multiple cloning site 5′ EcoRI cut site. TheP1 polypeptide sequence was derived from FMDV O1 Manisa serotype and wassynthesized by Genscript and inserted into the mpTarget vector followingdigestion of both the synthesized sequence and the mpTarget vector withMluI and NotI-HF restriction enzymes (New England Biolabs) as permanufacturer's instructions. Ligation, cloning and sequencing wasperformed as previously described herein.

Amplification of 3C from an FMDV Asia Lebanon 89 (Accession # AY593798,SEQ ID NO: 1) non-infectious template was performed using OneTaq 2×Master Mix with Standard Buffer (New England Biolabs) as permanufacturer's instructions and using primers NotI-3CLeb89-F (SEQ ID NO:173) and 3Casia-ns-EcoRI-R (SEQ ID NO: 184). The PCR product waspurified using a PCR purification kit (Qiagen). Both PCR product andmpTarget O1P1 plasmid were digested with NotI-HF and EcoRI-HFrestriction enzymes (New England Biolabs) as per manufacturer'sinstructions. Ligation, cloning and sequencing was performed aspreviously described.

Sequence for Δ1D2A-SGLuc was commercially synthesized (Genscript). Bothsynthesized sequence and the mpTarget O1P1-3C vector were digested withrestriction enzymes EcoRI-HF and XmaI (New England Biolabs) as permanufacturer's instructions. Ligation, cloning and sequencing wasperformed as described herein.

The nucleotide sequences of the O1P1-3C-SGLuc constructs are given bythe sequence identifier following each named construct:O1P1-3C(wt)-SGLuc (SEQ ID NO: 129), O1P1-3C(V28K)-SGLuc (SEQ ID NO:130), O1P1-3C(L127P)-SGLuc (SEQ ID NO: 131), O1P1-3C(V141T)-SGLuc (SEQID NO: 132), O1P1-3C(C142T)-SGLuc (SEQ ID NO: 133) andO1P1-3C(C163A)-SGLuc (SEQ ID NO: 134).

Example 8: Transformation and Harvesting of Cells Transfected withO1P1-3C-SGLuc Constructs

The transformation of HEK293-T cells with the prepared O1P1-3C-SGLucconstructs was performed as previously described herein. The incubationmedia was removed and assayed for luciferase activity as previouslydescribed herein. Cells were removed from surface by repeated pipettingof media then collected in a 15 ml conical tube (Falcon) and centrifugedat 500 rpm for 5 min to pellet cells. Cells were then re-suspended in200 μl of MPER mammalian protein extraction reagent (Invitrogen).Samples were mixed with 4× NuPAGE loading buffer (Invitrogen) permanufacturer's instructions and heated at 95° C. for 10 min. Sampleswere then briefly centrifuged and loaded onto NuPAGE Novex 4-12%Bis-Tris protein gels (Invitrogen) and run in 1×MES(2-(N-morpholino)ethanesulfonic acid) buffer at 200 V for 35 min. Theharvested, transformed cells were transferred to 0.2 μm pore size PVDFpre-cut blotting membranes (Invitrogen) with 1× transfer buffer(Invitrogen) per manufacturer's instructions.

Example 9: Western Blotting

Western blots were performed to examine for processed viral proteinsVP1-VP4. Three antibodies were used: (1) the F14 (Anti-VP0 and VP2)mouse monoclonal at 1:50 dilution, (2) the 12FE9 (Anti-VP1) mousemonoclonal at 1:50 dilution, (3) an Anti-VP3 rabbit polyclonal at 1:250dilution. All blots were performed with a one hour blocking step using5% milk followed by three five minute washes with 1×PBS-T (phosphatebuffered saline with Tween 20) buffer. Primary antibodies were appliedfor one hour at room temperature then removed and membranes washed threetimes for five minutes with 1×PBS-T buffer. Secondary antibodies usingeither Goat Anti-mouse-HRP (KPL) or Goat anti-rabbit-HRP (KPL) at 1:500dilutions were applied to membranes for one hour at room temperaturefollowed by three five-minute washes with 1×PBS-T. Visualization wasperformed using SIGMAFAST 3,3′-Diaminobenzidine tablets (Sigma) as permanufacturer's instructions for one hour at room temperature followed bytwo washes with double distilled water.

Example 10: Transmission Electron Microscopy (TEM)

Cells were grown in T-75 flasks for TEM. Cells were fixed in 2%glutaraldehyde in NaHCa (Heuser's) buffer, post-fixed with 1% tannicacid followed by 1% osmium tetroxide, en-bloc stained with 4% uranylacetate, embedded in 2% agarose, dehydrated through graded series ofacetone, and embedded in Spurr's resin (Electron Microscopy SciencesHatfield, Pa., USA). Ultrathin (80 nm) sections were cut on a Leica UC6,stained with uranyl acetate and lead citrate, and imaged on a Hitachi7600 with a 2 k×2 k AMT camera at 80 kV.

Example 11: Evaluation of Luciferase Activity of GLuc-3C Chimeras

A total of six GLuc-3C chimeras were evaluated for their luciferaseactivities: C163A, C142T, V141T, L127P, V28K, and wild-type. Assayreadings in FIG. 6 showed a diverse range outputs. The GLuc-3C chimeraexhibiting the highest luciferase output was the total activity knockoutmutant C163A while the lowest was the V141T mutation. As seen in FIG. 6,both the C142T and L127P mutations showed marked enhancement inluciferase outputs over the wild-type.

Example 12: O1P1-3C-SGLuc Chimeras

To check for processing of the P1 polypeptide, a second set ofconstructs containing the P1 polypeptide precursor derived from FMDV O1Manisa, followed by a nucleotide sequence encoding for a wild-type or amutant 3C protease with a C-terminal Δ1D2A sequence fused to it,followed by SGLuc, was constructed (O1P1-3C-SGLuc) (see for example,FIG. 7).

Assay readings of the O1P1-3C-SGLuc constructs of FIG. 8 showed asimilar distribution as those seen for the GLuc-3C constructs. The mostnotable difference is the heightened luciferase output of the L127Pconstruct which is now greater than seen with the C163A knockoutmutation.

The primary purpose of the O1P1-3C-SGLuc constructs was to evaluate theability of wild-type and mutant FMDV 3C proteases to process the P1polypeptide precursor in vivo (within the HEK293-T cells). To accomplishthis, transfected HEK293-T cell lysates were run on protein gels forwestern blots to detect fully processed viral capsid proteins VP1-VP4(see FIGS. 9A-9G). The only mutant to show no processing of the P1polypeptide was the complete activity knockout C163A. The two bandspresent with the anti-VP2 antibody represent the VP0 intermediate andthe fully processed VP2 polypeptide. Both bands are present in allsamples that show processing however the VP2 band is very weak in theV141T sample. Among all Western blot samples shown in FIGS. 9A-9G, it isclear that the L127P and C142T mutants possess the highest expressionwhile maintaining proteolytic activity towards the P1 polypeptideprecursor, based on the strong bands indicating VP2, VP3 and VP1presence as shown specifically in FIGS. 9D and 9F. FIG. 9A showing aWestern image of HEK293-T cells transfected with the Δ1D2A-SGLucconstruct of SEQ ID NO: 150 serves as a negative control to theanalysis.

Example 13: Formation of Virus-Like Particles (VLPs) in HEK293-T Cells

To examine for the presence of VLPs, transfected cell cultures wereexamined under transmission electron microscopy (TEM) for the presenceof arrays of empty capsids. Based on the luciferase readings and Westernblot analyses, HEK293-T cells transfected with O1P1-3C(wt)-SGLuc,O1P1-3C(C142T)-SGLuc, or O1P1-3C(L127P)-SGLuc were examined for VLPformation, to compare the two most promising mutations to the wild-type.Crystal arrays of FMDV VLPs were found in cells HEK293-T cellstransfected with O1P1-3C (wt)-SGLuc or O1P1-3C(C142T)-SGLuc, orO1P1-3C(L127P)-SGLuc, as seen in FIGS. 10A-B, FIGS. 11A-B, and FIG. 36Brespectively.

Example 14: Evaluation of the L127P/C142T Double Mutant

Combining the L127P and C142T mutations into a singleO1P1-3C(L127P/C142T)-SGLuc construct (SEQ ID NO: 135) dramaticallyenhanced transgene output when processing the P1 polypeptide of 01Manisa (SEQ ID NO: 102) (see FIG. 12), and results in an enhancementroughly 25× over the construct with the wild-type FMDV 3C protease. TheL127P/C142T construct retained the ability to process the P1 polypeptideprecursor, as seen in FIG. 13, as well as the ability to form arrays ofVLPs in transfected cell cultures, as shown in FIGS. 14A and 14B.

Example 15: Evaluation of FMDV 3C Protease Expression Towards E. coli

The FMDV 3C wild-type and mutant proteases (L127P, C142T, L127P/C142Tand C163A) were expressed in E. coli, and evaluated for the ability ofthe proteases to process the P1 polypeptide precursor in E. coli bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)analysis and their toxicity towards the bacterial host cells by cellgrowth assays in both plated, solid and liquid growth media havingkanamycin selection.

Accordingly, a construct containing the P1 polypeptide precursor derivedfrom FMDV O1 Manisa, was prepared as described herein and cloned intopET-SUMO vector (ThermoFisher Scientific) which had the SUMO tag removedprior to cloning of the P1 polypeptide. A construct encoding the FMDV 3Cwildtype and mutant proteases (L127P, C142T, L127P/C142T and C163A) witha N-terminal FLAG tag (SEQ ID NO: 190) was prepared as described hereinand cloned into pSNAP vector (New England Biolabs) according tomanufacturer's instructions.

The cell growth assays in plated and liquid growth media havingkanamycin selection indicate that the wild-type and mutant C142T FMDV 3Cproteases are toxic towards E. coli while the knock-out mutant C163AFMDV 3C protease and mutant L127P FMDV 3C protease are not toxic. Doublemutant L127P/C142T FMDV 3C protease appears to be more toxic than theC163A and L127P mutants but less toxic than the wild-type and the C142Tmutant (see FIGS. 15 and 16).

Further, as shown in FIG. 17, E. coli cells containing induced anduninduced P1 polypeptide precursor (Lanes 2 and 3), induced anduninduced mutant L127P FMDV 3C protease (Lanes 4 and 5), and induced anduninduced P1 polypeptide precursor and mutant L127P FMDV 3C protease(Lanes 6 and 7) are analyzed by western blotting with the F14(anti-VP0/VP2) mouse monoclonal antibody. FIG. 17 shows that the mutantL127P FMDV 3C protease retains its ability to process the P1 polypeptideprecursor in E. coli.

Overview of Examples 16-29

Monitoring protein expression in vitro and in vivo can pose significanthurdles. Current methods are typically antibody based methods such aswestern blotting or ELISA assays. For expression of transgene uponvaccination verification of expression requires testing for immuneresponse. This can cause complications during vaccine testing because alack of immune response does not absolutely indicate a failure of thevaccine to be expressed.

For in vitro work unless the protein of interest is secreted theseassays typically require the lysis of expressing cells in order toharvest expressed proteins. This can result in a need for multiplesamples which increases the number of variables in an experiment. Whendealing with transfected cell lines this can subject an experiment todifferent transfection efficiencies which can alter results. An idealsituation would be to link expression of a secreted easily detectablebiomarker with expression of any protein of interest. Such a systemwould allow for rapid determination of overall expression by monitoringcell culture media rather than needing to lyse cells. A similar systemin vivo using a biomarker that can be detected in the blood stream wouldgreatly aid in vaccine design and testing as it would allow forconfirmation of expression in an independent manner from immuneresponses.

Gaussia princeps luciferase (GLuc) is fast becoming a valuable moleculartool. GLuc is naturally secreted from expressing cells and capable of anintense luminescent burst while only being 185 amino acids in size. Ithas been used for in vivo work as GLuc is able to be detected in blood,plasma, and urine samples. The luminescent output of GLuc was enhancedthrough the mutation of two residues, F89W and I90L, resulting in asuper-luminescent 8990 GLuc (SGLuc) variant with a shifted peak emissionwavelength of 481 nm from 470 nm. If incorporated into a poly-cistronicexpression system the secreted nature of GLuc and subsequent GLucvariants, such as SGLuc, would make an excellent method to monitoroverall protein expression both in vitro and in vivo.

Polycistronic vectors have been created using several methods includingmultiple internal ribosome entry sites, proteolytic cleavage,self-processing peptides, and others. The Foot and Mouth Disease Virus(FMDV) 2A gene codes for a self-processing peptide that functions as atranslational interrupter separating the P1 and P2 regions of the FMDVpolypeptide in a non-proteolytic manner. The efficiency of 2A mediatedtranslational interruption is dependent upon the amino acid sequenceused. For the most efficient form of translational interruption to occuran additional sequence derived from the C-terminus of the VP1 proteinmust be present. Other 2A like sequences have been found including inthe other members of the Aphthovirus family, however the FMDV 2Asequence remains the most efficient at separation. Previous research hasincorporated 2A sequences to create poly-cistronic vectors capable ofexpressing multiple proteins for a wide range of uses.

Creation of a chimeric fusion between GLuc and 2A provides a mechanismby which protein expression could be easily monitored. In vitro, such achimera would confer the benefit of being able to monitor changes inprotein expression of transfected cells over time and provide a controlfor differences in transfection efficiency. This would greatly reduceexperimental variables and allow for more reliable time course studies.If incorporated into a vaccine construct, it could potentially allow fora direct correlation between expressions of vaccine peptides withluciferase activity.

In Examples 16-29, the creation and testing of six distinct chimerasbetween GLuc or SGLuc and the FMDV 2A translational interrupter sequenceare described. The effect of placement on the N- and C-terminus forGLuc/SGLuc on luciferase activity is evaluated. In addition, two DNAchimeras where the start codon of GLuc or SGLuc luciferase gene isdeleted, are created. The effect of the addition of a SGLuc chimera hason the creation of FMDV VLPs in cell culture is also evaluated.

It is found that regardless of whether or not the 2A sequence is on theN- or C-terminus, all six chimeras retained the ability to be secretedand to luminesce. Regardless of which terminus of GLuc/SGLuc the Δ1D2Asequence is added to, it does not fully inhibit either secretion orluminescence. However, placement of the Δ1D2A on the C-terminus ofGLuc/SGLuc does have a notable impact on luminescence but is stillreadily detectable in cell culture. The presence of Δ1D2A on theN-terminus of GLuc or SGLuc allows for the deletion of the methionine atposition one of the GLuc or SGLuc genes without major deleterious effectto either luminescence or secretion. Finally, the present inventorssurprisingly found that when such a chimera is added to the C-terminusof a vaccine construct capable of producing FMDV VLPs, it allows forverification of protein expression and the formation of VLPs in vitro.

GLuc or SGLuc chimeras are useful in vaccine design and development.When testing new vaccines, it is often unknown if the vaccine failed toproduce the transgenic product in the test subject or if the immunesystem of the test subject failed to respond to transgene produced bythe vaccine. The inventors have surprisingly discovered that embodimentsof GLuc or SGLuc chimeras of the present invention provide a means toconfirm protein production from the vaccine in vivo.

Example 16: Insertion of GLuc into pTarget and Cloning of H3 intopTarget

Gaussia luciferase (GLuc) template sequence was inserted in the pTargetvector as described in Example 1 to form pTarget-GLuc.

To clone Bovine Histone H3 into pTarget, cDNA synthesized from bovineRNA was utilized as a template. PCR was performed as per manufacturer'sinstructions using OneTaq 2× Master Mix with Standard Buffer (NewEngland Biolabs) with primers H3-F (SEQ ID NO: 175) and H3-R (SEQ ID NO:176). Sequencing to confirm insertion of Histone H3 coding sequence intothe pTarget vector was performed by sequencing with primers T7 (SEQ IDNO: 179) and Seq-R (SEQ ID NO: 180) and analysis of sequencing data wasanalyzed by Sequencher 4.8 program (Genecodes).

Example 17: Site Directed Mutagenesis of GLuc

Site directed mutagenesis of GLuc was performed using the GENEART®Site-Directed Mutagenesis System (Invitrogen) as per manufacturer'sinstructions with primers SGLuc8990-MF (SEQ ID NO: 177) and SGLuc8990-MR(SEQ ID NO: 178). Confirmation of mutation was performed by sequencingwith primers T7 (SEQ ID NO: 179) and Seq-R (SEQ ID NO: 180) and analysisof sequencing data was analyzed by Sequencer 4.8 program (Genecodes).

Example 18: Construction of GLuc/SGLuc-Δ1D2A Chimeras

For the construction of GLuc-Δ1D2A chimera, PCR amplification wasperformed with pTarget-GLuc as a template using OneTaq 2× Master Mixwith Standard Buffer (New England Biolabs) and primers T7 (SEQ ID NO:179) and GLuc-NS-NheI-R (SEQ ID NO: 181) per manufacturer'sinstructions. PCR product was digested with NheI-HF and XhoI (NewEngland Biolabs) restriction enzymes as per manufacturer's instructions.

A construct containing the Δ1D2A sequence in a pCRII vector was used asa cloning template for construction of GLuc-Δ1D2A. The vector wasdigested with NheI-HF and XhoI restriction enzymes (New England Biolabs)as per manufacturer's instructions. Ligation of digested GLuc sequenceinto digested pCRII vector was performed using T4 DNA ligase (Roche) asper manufacturer's instructions. Creation of the GLuc-Δ1D2A chimera inthe pCRII vector was confirmed by sequencing with T7 (SEQ ID NO: 179)and GLuc-NS-NheI-R (SEQ ID NO: 181) and analysis of sequencing data wasanalyzed by Sequencher 4.8 program (Genecodes).

For the insertion of GLuc-Δ1D2A into pTarget plasmid, PCR amplificationwas performed with pCRII GLuc-Δ1D2A as a template and using OneTaq 2×Master Mix with Standard Buffer (New England Biolabs) and primersAscI-Kzk-Gluc-F (SEQ ID NO: 182) and 2A-XmaI-R (SEQ ID NO: 196).Confirmation of insertion was performed by sequencing with primers T7(SEQ ID NO: 179) and Seq-R (SEQ ID NO: 180) and analysis of sequencingdata was analyzed by Sequencher 4.8 program (Genecodes).

To create SGLuc-Δ1D2A, site directed mutagenesis was performed using theGENEART® Site-Directed Mutagenesis System (Invitrogen) as permanufacturer's instructions with primers SGLuc8990-MF (SEQ ID NO: 177)and SGLuc8990-MR (SEQ ID NO: 178) and using pTarget Gluc-Δ1D2A as atemplate. Confirmation of mutation was performed by sequencing withprimers T7 (SEQ ID NO: 179) and Seq-R (SEQ ID NO: 180) and analysis ofsequencing data was analyzed by Sequencher 4.8 program (Genecodes).

Example 19: Construction of Δ1D2A-GLuc/SGLuc Chimeras

For the construction of Δ1D2A-GLuc/SGLuc chimeras, a nucleotide sequenceencoding the Δ1D2A-SGLuc sequence was synthesized by Genescript in thepUC57 kan vector. PCR amplification was performed using OneTaq 2× MasterMix with Standard Buffer (New England Biolabs) and primers AscI-Kzk-2A-F(SEQ ID NO: 185) and Gluc-R-NotI (SEQ ID NO: 186) per manufacturer'sinstructions. Insertion into the pTarget vector (Promega) followedmanufacturer's instructions for T/A cloning. Confirmation of insertionwas performed by sequencing with primers T7 (SEQ ID NO: 179) and Seq-R(SEQ ID NO: 180) and analysis of sequencing data was analyzed bySequencher 4.8 program (Genecodes).

To construct the Δ1D2A-Gluc chimera the pTarget Δ1D2A-SGLuc constructwas used as a template for site directed mutagenesis using the GENEARTSite-Directed Mutagenesis System (Invitrogen) as per manufacturer'sinstructions with primers Gluc8990-MF-2 (SEQ ID NO: 197) andGluc8990-MR-2 (SEQ ID NO: 198). Confirmation of mutation was performedby sequencing with primers T7 (SEQ ID NO: 179) and Seq-R (SEQ ID NO:180) and analysis of sequencing data was analyzed by Sequencher 4.8program (Genecodes).

Example 20: Construction of Δ1D2A-GLuc Δ1M (SEQ ID NO: 151) andΔ1D2A-SGLuc Δ1M Chimeras (SEQ ID NO: 152)

To construct Δ1D2A-Gluc/SGLuc Δ1M chimeras the appropriate pTargetΔ1D2A-GLuc or pTarget Δ1D2A-SGLuc construct was used as a template forsite directed mutagenesis using the GENEART Site-Directed MutagenesisSystem (Invitrogen) as per manufacturer's instructions with primers NoMet Gluc-MF (SEQ ID NO: 187) and No Met Gluc-MR (SEQ ID NO: 188).Confirmation of mutation was performed by sequencing with primers T7(SEQ ID NO: 179) and Seq-R (SEQ ID NO: 180) and analysis of sequencingdata was analyzed by Sequencher 4.8 program (Genecodes).

Example 21: Construction of P1-3C and P1-3C-SGLuc Constructs

The nucleotide sequence derived from FMDV O1 Manisa serotype and codingfor the P1 polypeptide (SEQ ID NO: 136) was synthesized by Genscript.Nucleotide sequence coding for the P1 polypeptide was cloned into amodified pTarget vector using BamHI-HF and NotI-HF restriction enzymes(New England Biolabs) as per manufacturer's instructions. For thepreparation of the P1-3C construct, insertion of 3C was performed byusing PCR amplification with primer NotI-3CLeb89-F (SEQ ID NO: 173) andprimer 3CLeb89-EcoRI-R (SEQ ID NO: 199). For the preparation of theP1-3C-SGLuc construct, insertion of 3C was performed by using PCRamplification with primers NotI-3CLeb89-F (SEQ ID NO: 173) and3Casia-ns-EcoRI-R (SEQ ID NO: 184). The sequence for 2A-SGLuc was theninserted behind the 3C sequence by digestion of the synthesized templateprovided by Genscript with EcoRI-HF and XmaI restriction enzymes (NewEngland Biolabs) as per manufacturer's instructions. All ligations wereperformed as previously described herein.

Example 22: Transfection and Harvesting of HEK293-T Cells

HEK293-T cell line was a generous gift from USDA-ARS-FADRU. Cells weregrown in 1×MEM media (Gibco) with 10% Fetal Bovine Serum defined(HyClone), 1× Antibiotic-Antimycotic (Gibco), 1×MEM-NEAA (non-essentialamino acids) (Gibco), and 1× Glutamax (Gibco). Cells were transfected atpassage 58 in a six-well plate using Lipofectamine 2000 (Invitrogen) asper manufacturer's instructions. After incubating for 24 hours in a 37°C. CO₂ incubator, media from transfected cells was removed and stored ina 2 mL tube at 4° C. To harvest cell lysates 200 μl of 2× LuciferaseCell Lysis Buffer (ThermoFisher Scientific) was added to each well andpipetted to remove cells attached to the plate. Cell lysates were put ina 1.5 ml tube and stored at −70° C.

Example 23: Luciferase Assay

Luciferase activity was measured using a 96-well BioSystems Veritasluminometer (Turner Biosystems). For unadjusted samples, 20 μl ofharvested media was used and readings taken with no delay after aninjection of 25 μl of 100 μM water soluble coelenterazine solution(NanoLight Technologies, Pinetop Ariz.). An integration time of 0.5 swas used for data collection both before and after injection ofcoelenterazine. Readings for pre-injection were used to establish abaseline of light emission at the time of injection and subsequentlysubtracted from the post-injection values during data analysis.Replicates were averaged together to give relative light units per halfsecond (RLU/0.5 s).

To adjust for differential expression of transgene amongst samplesharvested media was mixed with 4× NuPage LDS Sample Buffer (Invitrogen),heated at 97° C. for 10 minutes, then loaded into wells on 10-wellNuPage 4-12% Bis-Tris gels (Invitrogen). Gels were run in 1×MES buffer(Invitrogen) at 200 V for 35 minutes. Samples were then transferred ontomembranes using the i-Blot system (Invitrogen). Membranes were incubatedin 5% milk blocking buffer for 40 minutes then washed three times with1×PBS-T buffer (EMD Millipore) at 5 minutes each. A 1:1000 dilution ofRabbit polyclonal Antisera-Gluc (NanoLight Technologies, Pinetop Ariz.)was used for primary antibody incubation while shaking at roomtemperature for 1 hour. Three washes were repeated with 1×PBS-T for 5minutes each after primary antibody incubation. A 1:500 dilution of goatanti-rabbit-HRP secondary antibody was then applied and allowed toincubate while shaking at room temperature for 1 hour followed again bythree washes with 1×PBS-T for 5 minutes each. DAB staining was performedusing SIGMAFAST 3,3′-Diaminobenzidine tablets (Sigma) dissolved in 15 mLof ddH2O for 1 hour followed by de-staining with two rounds of washingwith 1×PBS-T for 5 minutes. Volumes of media loaded onto the gel wereadjusted until equal loading was obtained for each sample.

Luciferase assay on equilibrated samples was performed as describedabove with the only difference being the usage of adjusted volumes ofcell culture media determined by analysis of western blots as a sample.Media from untransfected HEK293-T cells was used to dilute samples inorder to maintain a constant volume.

To measure the effects of cell lysis buffers on luminescence 10 μlharvested GLuc and SGLuc media was mixed with 90 μl of either cellculture media, 2× Luciferase Cell Lysis Buffer (ThermoFisher), or MPER(Invitrogen). A total of 100 μl was used in each well with no delayafter an injection of 25 μl of 50 μM water soluble coelenterazinesolution (NanoLight Technologies, Pinetop Ariz.). An integration time of0.5 seconds was used for data collection both before and after injectionof coelenterazine.

Example 24: Transfection of LF-BK αV/P6 Cells

The LF-BK αV/β6 cell line was grown in 1×DMEM media (Gibco) with 10%Fetal Bovine Serum defined (HyClone), 1× Antibiotic-Antimycotic (Gibco),1×MEM-NEAA (Gibco), and 1× Glutamax (Gibco). Cells were transfected atpassage 44 in a six well plate using Lipofectamine 2000 (Invitrogen) asper manufacturer's instructions. After incubating for 24 hours in a 37°C. CO₂ incubator media from transfected cells was removed and used forluciferase assays as described herein.

Example 25: Immunofluorescence Assay and Immune Electron Microscopy

The LF-BK αV/β6 cell line was grown in T-75 flasks for IFA andimmunoelectron microscopy (I-EM). IFAs were performed using threedifferent antibodies 6HC4, 12FE9, and F21. The 6HC4 antibody is specificto FMDV serotypes other than O when used in IFA and was used as anegative control. Antibodies 12FE9 and F21 are specific to FMDV type OVP1 and all FMDV serotype VP2 peptides, respectively. For I-EM samplesare fixed in 4% paraformaldehyde with periodate and lysine in sodiumcacodylate buffer, embedded in 2% agarose, partially dehydrated inethanol, embedded in medium grade LR White resin (Electron MicroscopySciences). Ultrathin (80 nm) sections were cut on a Leica UC6.Immunohistochemistry was performed with antibody F21 at a 1:10 dilutionand goat anti-mouse ultrasmall nanogold (Electron Microscopy Sciences),enhanced with GoldEnhance EM (Nanoprobes), post stained with uranylacetate, and imaged on a Hitachi 7600 with a 2 k×2 k AMT camera at 80kV.

Example 26: Summary of GLuc or SGLuc Δ1D2A Chimeras Constructed

Eight different constructs were used to evaluate the effect of addingthe FMDV 2A gene to GLuc/SGLuc in the present disclosure, as shown inFIG. 18.

The nucleotide sequence of construct GLuc is SEQ ID NO: 145 or 200.

The nucleotide sequence of construct SGLuc is SEQ ID NO: 146 or 202.

The nucleotide sequence of construct GLuc-Δ1D2A is SEQ ID NO: 147.

The nucleotide sequence of construct SGLuc-Δ1D2A is SEQ ID NO: 148.

The nucleotide sequence of construct Δ1D2A-GLuc is SEQ ID NO: 149.

The nucleotide sequence of construct Δ1D2A-SGLuc is SEQ ID NO: 150.

The nucleotide sequence of construct Δ1D2A-GLuc Δ1M is SEQ ID NO: 151.

The nucleotide sequence of construct Δ1D2A-SGLuc Δ1M is SEQ ID NO: 152.

Constructs GLuc-Δ1D2A, SGLuc-Δ1D2A, Δ1D2A-GLuc, Δ1D2A-SGLuc, Δ1D2A-GLucΔ1M, and Δ1D2A-SGLuc Δ1M, are chimeras between GLuc or SGLuc and theFMDV 2A translational interrupter. The remaining two constructs wereunaltered GLuc and SGLuc used as controls. To facilitate efficientseparation, a modified 2A sequence identified as Δ1D2A, as shown in FIG.4A, was utilized. This modified sequence is derived from the FMDV A24virus and contains the truncated 11 C-terminal amino acids of VP1 (Δ1D),the defined 2A sequence, and the N-terminal proline (+1 Proline)required for highly efficient translational interruption. ConstructsΔ1D2A-GLuc Δ1M and Δ1D2A-SGLuc Δ1M have the methionine at amino acidposition one in the Gluc and SGLuc genes respectively deleted since itis no longer needed for translation initiation.

Example 27: Secretion and Luminescence of Constructs

A concern when creating chimeras between GLuc/SGLuc and Δ1D2A was thatthe addition of the Δ1D2A sequence on either the N- or C-terminus mightprevent secretion of the luciferase. Supernatant was taken off oftransfected cell cultures and the supernatant was examined forluciferase activity. FIG. 19 shows that luciferase activity was found inall samples except the negative control confirming that addition of theΔ1D2A sequence on either the N- or C-terminus does not prevent secretionor luminescence. This data did show a peculiarity in that the additionof the Δ1D2A sequence on either the N- or C-terminus of GLuc appeared toenhance luminescence output often in excess of similar SGLuc chimeras.

A number of variables independent of enzyme activity could be capable ofaltering luciferase readings from transfected cell media. Minorvariations in cell confluence, transfection efficiency, or minor genomicDNA contamination in plasmid preps can lead to alterations in proteinexpression that may give altered readings amongst samples. To accountfor these variables, the harvested media was taken off of transfectedcell cultures and equal loading was determined by western blotting.Western blots shown in FIG. 20A confirm the 2A induced separation inN-terminal chimeras and the subsequent increase in molecular weightcaused by the presence of Δ1D2A in C-terminal chimeras. Luminescentoutputs were compared at the roughly equal protein concentrations asdetermined by western blots shown in FIG. 20A (see FIG. 20B).

FIG. 20B show that chimeras with Δ1D2A on the C-terminus displayedreduced luminescent outputs when compared to unmodified GLuc or SGLucand N-terminal chimeras. The deletion of the first methionine, Δ1M, fromN-terminal chimeras had little to no effect on luminescent output nordid it prevent subsequent secretion.

An interesting observation was that despite previous reports identifyingSGLuc variant as emitting roughly 10× stronger bioluminescence, only amoderate increase in bioluminescence when concentrations wereequilibrated by western blotting was observed. Addition of Δ1D2A toeither terminus negated any difference in luminescent output betweenGLuc and SGLuc.

Example 28: Changes in Luminescence in the Presence of Cell LysisBuffers Induced by the 8990 Mutation

Previous reports have identified two mutations at residues 89 and 90that produce a super-luminescent GLuc variant, known as SGLuc that alsoshifted the peak luminescence from 470 nm to 481 nm. The previous studyutilized secretion deficient mutants and lysed the cells to harvest theluciferase.

The mutations at residues 89 and 90 of GLuc also positively affect theability of SGLuc to luminesce in cell lysis buffers. The data in FIG. 21shows that there is a noticeable drop in the percentage of luciferaseactivity retained when mixed with cell lysis buffers using GLuc. Thisdrop is lessened when using the SGLuc variant. Luciferase activity isreduced more than 80% when GLuc is mixed with cell lysis bufferscompared to a reduction of less than 40% when using the SGLuc variant.This supports the conclusion that the mutations that make the SGLucvariant enhance luminescence in cell lysis buffer.

It was found in Examples 26-28 that the Δ1D2A sequence can be linked toGLuc/SGLuc in either an N- or C-terminal manner without preventingeither secretion or luminescent output (see FIG. 19). The differences inluminescent output between FIG. 19 and FIG. 20B demonstrate why a systemsuch as described here is invaluable. When adjusted to equalize proteinlevels in the luminescent assay of FIG. 20B, the data looks dramaticallydifferent than when simply examining harvested media (see FIG. 19). Thisis due to any one of a number of variables that can influence the amountof protein produced following transfection. Minimization of thesevariables is useful for better interpretation of experimental results.The chimeras described in the present disclosure provide a means bywhich these variables can be minimized and/or accounted for by usingluminescent activity as a control.

The data presented in FIG. 20B shows that there is a notable differencein luminescent output between N- and C-terminal GLuc/SGLuc chimeras. Theaddition of the Δ1D2A sequence on the C-terminus results in a largersecreted protein and lowered luminescent output, as seen in FIGS. 20Aand 20B. The addition of an N-terminal Δ1D2 Å does not appear to have aslarge of an impact on luminescent output although there is a drop whenusing the SGLuc variant.

One of the most unexpected findings in Examples 26-28 is that when usingan N-terminal GLuc/SGLuc chimera the start codon (methionine) of theGLuc/SGLuc gene can be deleted without any major deleterious effects onluciferase activity or secretion (see FIGS. 19, 20A-20B, 21). In anon-chimeric protein removal of the methionine at position 1 of thestart codon of an amino acid sequence requires the use of a form ofpost-translational protein processing. By creating a chimera with theΔ1D2A sequence this methionine can be effectively mutated to a proline.Separation with the N-terminal sequence is highly efficient, as there isno need for post-translation protein processing, and there are nointermediates still containing the first methionine present. Theunexpected findings in Examples 26-28 demonstrate that usage of theΔ1D2A sequence can provide a valuable tool in molecular biology toexamine structural and functional influences of the methionine coded forby the start codon.

Example 29: Viability of a Marker for Expression of FMD VaccineConstructs

In order to ensure that the presence of the luciferase did notnegatively impact the ability of an FMDV construct to form virus likeparticles (VLPs), a plasmid vector carrying one of the three constructsset forth in FIG. 22 was included in transfected cell cultures. Aplasmid encoding SGLuc alone “mpTarget SGLuc” construct (SEQ ID NO: 153)was used as a control for the experiment and the Δ1D2A-SGLuc was placedon the C-terminus of an FMDV construct capable of creating VLPs usingthe FMDV O1 Manisa serotype as a template for the P1 polypeptide. Mediafrom transfected cells was tested for luciferase activity. As seen inFIG. 23, no luciferase activity was detected in the P1-3C construct,“mpTarget P1-3C” construct (SEQ ID NO: 155), which did not have theSGLuc gene. Luciferase activity was detected in both cells transfectedwith SGLuc only and the P1-3C-Δ1D2A-SGLuc construct, “mpTargetP1-3C-Δ1D2A-SGLuc” construct (SEQ ID NO: 154). Luminescent output of theconstruct with Δ1D2A-SGLuc was significantly higher than backgroundluminescent levels, as seen in FIG. 23.

Immunofluorescence assays (IFA) were performed to confirm expression ofFMDV peptides in transfected cells, using three different antibodies6HC4, 12FE9, and F21, as shown in FIGS. 24A-24I. The 6HC4 antibody isnon-reactive with FMDV type 0 serotype when used in IFA and was used asa negative control. Antibodies 12FE9 and F21 are reactive to FMDV type01 Manisa VP1 and VP2 peptides respectively.

A minor level of background activity was seen in all three samples withthe 6HC4 antibody. No significant reactivity with either 12FE9 or F21was seen in the cells transfected with plasmid containing only SGLuc(see FIG. 24D and FIG. 24G respectively). Reactivity with only 12FE9 andF21 were seen in both the P1-3C and the P1-3C-Δ1D2A-SGLuc samples (seeFIGS. 24E, 24F, 24H, and 24I). This confirms that FMDV O1 Manisa capsidproteins were being expressed in transfected cell cultures.

Confirmation of FMDV expression in transfected cells by IFA does notconfirm VLP formation. Therefore, to evaluate whether or not VLPformation was being impaired, immune-electron microscopy (I-EM) wasperformed on P1-3C and the P1-3C-Δ1D2A-SGLuc samples, as shown in FIGS.25A-25D. Previous reports in the literature indicate that when FMDV isexamined under electron microscopy, crystalline arrays of capsids areobserved. Such similar structures in both cells transfected with theP1-3C-Δ1D2A-SGLuc and P1-3C plasmids indicative of VLP formation in bothsamples in FIGS. 25A-25B and FIGS. 25C-25D, respectively.

Hence, the presence of the Δ1D2A-SGLuc in the construct did not preventthe expression, (as shown in FIGS. 24A-24I), or function, (as shown inFIGS. 25A-25D), of the FMDV peptides in the plasmid construct. Theconstruction of VLPs in cell culture, as shown in FIGS. 25A-25D, is aparticularly critical step in FMD vaccine development as it shows theability to construct structures akin to those seen in normal FMDVinfection.

Example 30: Mechanism for Enhancement of Transgene Output by Mutant FMDV3C Proteases

The wild-type FMDV 3C protease has been shown to induce proteolyticcleavage of several host proteins, including but not limited to histoneH3, nuclear transcription factor kappa B essential modulator (NEMO),Src-associated substrate in mitosis of 68 kDa (SAM68), eukaryotictranslation initiation factor 4A1 (eIF4A1), and eukaryotic translationinitiation factor 4G (eIF4G).

During infection with FMDV, the FMDV 3C protease processes the hosteIF4AI protein causing disruption to host translational machinery.Subsequently when 3C is included in vaccine constructs it results in asimilar processing of host eIF4AI which has negative implications oncellular function. To evaluate a potential mechanism for enhancement oftransgene output by mutant FMDV 3C proteases, an examination of theirability to process bovine eIF4AI was performed using a cell free system.

Plasmids were constructed using the pSNAP vector to express bovineeIF4AI and the individual 3C mutants. These plasmids were then used toevaluate for processing of eIF4AI in the cell free system, as shown inFIG. 26. These data show that any construct which contains the L127Pmutation has significantly lowered processing of host eIF4AI whencompared to other 3C mutants. This provides a mechanism by which theL127P mutation, either by itself or in conjunction with the C142Tmutation, is able to enhance transgene output in transfected cell.

By preventing the processing of host eIF4AI 3C constructs containing theL127P mutation are able to prevent a significant negative consequence ofFMDV 3C protease expression. This results in a more normal cellularfunction of cells expressing vaccine constructs which enhances the totalamount of transgene product that can be produced.

Overview of Examples 31-34

The data presented in Examples 31-34 confirm that when the serotype O P1polypeptide previously used to evaluate 3C mutants for processing andtoxicity is replaced with a P1 derived from type SAT2 the FMDV 3Cprotease mutants retain the ability to process the P1 and the processedVPs retain the ability to form VLPs. Since the 3C region of FMDVserotypes is strongly conserved, this increases the likelihood that 3Cmutations based on the Asia Lebanon 89 template will be able to processP1s from multiple serotypes and produce assembled VLPs.

Example 31: Construction of Vectors Containing SAT2P1, 3C and 2A-SGLucReporter (mpTarget-SAT2P1-3C-2ASGLuc)

Constructs containing a P1 polypeptide derived from the SAT2 Egypt 2010strain were created to evaluate for transgene expression, polypeptideprocessing, and VLP formation when using Asia Lebanon 89 wildtype 3C andderived mutants L127P, C142T, C163A, as well as the L127P/C142T doublemutant. Constructs were constructed in the mpTarget vector and containsthe P1 polypeptide, 3C protease, and a 2A-SGLuc luciferase reporter tomonitor transgene output, as shown in FIG. 27.

Example 32: Evaluation of Transgene Expression ofmpTarget-SAT2P1-3C-2ASGLuc Constructs

HEK293-T cells were transfected with each of the constructs and mediamonitored for luciferase activity, as shown in FIG. 28. From this datain FIG. 28, it can be observed that while the C142T mutation does notenhance transgene output as much as previously observed with the O1Manisa P1 constructs while the L127P single mutation and L127P/C142Tdouble mutation do retain a significant enhancement of transgene outputover the wild-type FMDV 3C protease. The presence of the double mutationalso continues to enhance transgene output over the single L127Pmutation.

Example 33: Evaluation of P1 Processing of mpTarget-SAT2P1-3C-2ASGLucConstructs

While retention of enhanced transgene output is important what is morecritical is the ability of these mutations to process the SAT2 P1polypeptide and assemble virus like particles (VLPs). To accomplish thiscell lysates of HEK293-T cells transfected with each construct were runon a protein gel and examined by western blotting. In FIGS. 29A-29C,processing of the P1 by all mutants is present with the exception of theC163A complete activity knockout. While some unprocessed P1 is retainedin the L127P and L127P/C142T samples a significant amount of fullyprocessed VPs are observed. This confirms that both mutations retain theability to process a SAT2 P1 which has a divergent amino acid sequencefrom the type 0 serotype previously used. The lack of clear and distinctVP1 and fully processed VP2 bands in wild-type and C142T is probably dueto low concentration of sample loaded onto the gel.

Example 34: Evaluation of VLP Formation of mpTarget-SAT2P1-3C-2ASGLucConstructs

Processing of the P1 polypeptide does not guarantee the formation ofVLPs. To assess the retention of SAT2 P1 containing constructs to formVLPs, transfected cell cultures were examined using electron microscopy.In FIGS. 30A-30F, VLP arrays in three of the four samples that showed P1processing can be observed. Ironically the only sample not to displayVLP arrays was the wild-type 3C sample (not shown). These data confirmthat the presence of the L127P, C142T, or the L127P/C142T doublemutation does not prevent the formation of VLPs from a processed SAT2 P1polypeptide. Previous to this work, VLP arrays had only been observedwith type 0 serotypes of FMDV. The results shown in FIGS. 30A-30Frepresent the first observance of VLP arrays using a P1 of any FMDVserotype other than type O.

Example 35: Electronmicroscopic Confirmation of VLP Formation inBacteria

A two plasmid system was used to produce VLPs in bacteria. The twoplasmids utilized were pET His-01P1 (SEQ ID NO: 219) and pSNAPFlag-3C(L127P) (SEQ ID NO: 220). In this system the FMDV P1 was encodedin a first plasmid while the FMDV 3Cpro sequence was encoded in a secondplasmid. The pET His-01P1 plasmid confers the ability for bacteria togrow in the presence of kanamycin while the pSNAP Flag-3C(L127P) plasmidconfers the ability for bacteria to grow in the presence of ampicillin.By transforming a single bacteria with both plasmids, the bacteria isable to grow in the presence of kanamycin and ampicillin ensuring thatonly bacteria with both plasmids present are able to be propagated.

Bacteria were transformed with either both plasmids, pET His-O1P1/pSNAPFlag-3C(L127P), or the single pET His-O1P1. Protein expression was theninduced by the addition of 1 μM IPTG. Bacteria cells were then lysedwith B-PER and loaded onto a protein gel to check for expression andprocessing by utilizing western blotting, as shown in FIG. 31.

FIG. 31 shows a definitive band representing VP1 in the pETHis-O1P1/pSNAP Flag-3C(L127P) sample and a lack of the band in the pETHis-O1P1 sample which lacks any protease to process it. As expected,both samples showed the presence of the P1 precursor polypeptide.Additionally, a 1CD fusion band in the pET His-O1P1/pSNAP Flag-3C(L127P)was present in the sample, indicating of the presence of partiallyprocessed products.

Samples of the bacterial lysate were then used for aco-immunoprecipitation assay using either the B473M antibody (ABCAM) orthe 12FE9 antibody as shown by FIG. 32. The co-IPs with B473M antibodyshowed a weak pull down of 1ABC, VP0, VP1 and VP2, and a definitive pulldown of VP3. The pull down with 12FE9 which recognizes FMDV VP1, shows adefinitive capture of 1ABC, VP0, VP2, VP3, and VP1.

The presence of VP2 suggested that VLPs were forming. To evaluate thisbacteria transformed with pET His-01P1 and pSNAP Flag-3C(L127P) wereevaluated by electron microscopy, FIGS. 33A-B. Examination oftransformed bacteria by EM confirmed the presence of VLPs beingproduced. VLPs have immunogenic properties absent from individual virusproteins making them good candidates for a new FMDV vaccine.

Example 36: Expression and Processing of FMDV P1 Precursor Polypeptidein SF21 Cells

Baculovirus vectors encoding O1P1 FMDV 3C protease (L127P) or FMDV 3Cprotease (L127P/C142T)-SGLuc were transformed into Spodoptera frugiperdaSF21 cells. SF21 cells were cultured and supernatants were recovered,resolved by Western blotting, and probed with F14 (mouse monoclonalantibody to VP0/VP2), anti-VP3 rabbit polyclonal antibody, and 12FE9(mouse monoclonal antibody to VP1). Both the L127P and L127P/C142T 3Cprotease mutants exhibited proteolytic activity on FMDV P1 protein.

The FMDV VP0 protein is around 33 kDa in molecular weight and VP2, VP3and VP1 each have a molecular weight of about 24 kDa.

As apparent from the F14 (anti-VP0/VP2) Western blots in FIG. 34, VP0was processed into smaller about 24 kDa subunits. The anti-VP3 Westernblots in FIG. 34 show banding at about 24 kDa which is consistent withprocessing and cleavage of VP3 from the longer P1 precursor polypeptide.The 12FE9 (anti-VP1) Western blots in FIG. 34 show discrete bands above28 kDa which is slightly more than the approximate molecular 24 kDamolecular weight of VP1.

Example 37: 3C(L127P)-SGLuc Chimeras Containing the P1 PolypeptidePrecursor from Various FMDV Serotypes

Constructs containing the P1 polypeptide precursor derived from fivedifferent FMDV serotypes—O1 Manisa (SEQ ID NO: 136), A24 Cruzeiro (SEQID NO: 138), Asial Shamir (SEQ ID NO: 144), C3 Indrial (SEQ ID NO: 141),and SAT2 Egypt (SEQ ID NO: 140), each followed by a nucleotide sequenceencoding for a modified 3C protease at L127P (SEQ ID NO: 141) with aC-terminal Δ1D2A sequence fused to it, followed by SGLuc, wereconstructed.

These five constructs were evaluated on the ability of the FMDV 3Cproteases modified at L127P to process the P1 polypeptide precursor invivo (within the HEK293-T cells). To accomplish this, transfectedHEK293-T cell lysates were run on protein gels for western blots todetect fully processed viral capsid proteins VP1-VP4 (see FIGS. 35A-D)utilizing the F14 (anti-VP0/2), anti-VP3, 6HC4 (anti-VP1), and 12FE9(anti-VP1) antibodies.

The FMDV VP0 protein is around 33 kDa in molecular weight and VP2, VP3and VP1 each have a molecular weight of about 24 kDa.

As apparent from the F14 (anti-VP0/VP2) Western blots in FIG. 35A, VP0was processed into smaller 24 kDa subunits. The anti-VP3 Western blotsin FIG. 35B show banding at about 24 kDa which is consistent withprocessing and cleavage of VP3 from the longer P1 precursor polypeptide.The 6HC4 (anti-VP1) Western blots in FIG. 35C show banding that areslightly below 28 kDa, and around the 24 kDa molecular weight of VP1.The 12FE9 (anti-VP1) Western blot in FIG. 35D shows a band for thelysates of the HEK293-T cells transfected with the O1P1-3C(L127P)-SGLucthat is slightly below 28 kDa which is around the 24 kDa molecularweight of VP1.

The Western blot samples show that the 3C(L127P) mutant retainsproteolytic activity towards the P1 polypeptide precursor among the fivedifferent FMDV serotypes, based on the bands indicating VP2, VP3 and VP1presence as shown specifically in FIGS. 35A-D.

Example 38: Evaluation of VLP Formation ofmpTarget-AsiaP1-3C(L127P)-SGLuc Construct

Processing of the P1 polypeptide does not guarantee the formation ofVLPs. To assess the retention of Asia P1 containing constructs to formVLPs, transfected cell cultures were examined using electron microscopy.In FIG. 36A, VLP arrays show that an Asia P1 processed by 3C(L127P)retains the ability to form VLPs.

The foregoing discussion discloses embodiments in accordance with thepresent disclosure. As will be understood by those skilled in the art,the approaches, methods, techniques, materials, devices, and so forthdisclosed herein may be embodied in additional embodiments as understoodby those of skill in the art, it is the intention of this application toencompass and include such variation. Accordingly, this disclosure isillustrative and should not be taken as limiting the scope of thefollowing claims.

What is claimed:
 1. A DNA vaccine against foot-and-mouth disease virus(FMDV), comprising: a polynucleotide encoding a FMDV P1 precursorpolypeptide; and a polynucleotide encoding a modified FMDV 3C protease,wherein the modified FMDV 3C protease comprises a L127 amino acidsubstitution of a wild-type FMDV 3C protease.
 2. The DNA vaccine ofclaim 1, wherein the polynucleotide encoding a FMDV P1 precursorpolypeptide and the polynucleotide encoding a modified FMDV 3C proteaseare contained in a single vector or polynucleotide construct.
 3. The DNAvaccine of claim 1, comprising a first vector or polynucleotideconstruct comprising the polynucleotide encoding a FMDV P1 precursorpolypeptide, and a second vector or polynucleotide construct comprisingthe polynucleotide encoding a modified FMDV 3C protease.
 4. The DNAvaccine of claim 1, comprising a polynucleotide encoding a fusionprotein of the modified FMDV 3C protease and the FMDV P1 precursorpolypeptide.
 5. The DNA vaccine of claim 1, wherein the polynucleotideencoding a FMDV P1 precursor polypeptide and the polynucleotide encodinga modified FMDV 3C protease together comprise a single open readingframe encoding both the FMDV P1 precursor polypeptide and the modifiedFMDV 3C protease.
 6. The DNA vaccine of claim 1, formulated against atleast one of FMDV 0 serotype, A serotype, C serotype, Asia 1 serotype,SAT1 serotype, SAT2 serotype or SAT3 serotype.
 7. The DNA vaccine ofclaim 1, formulated against two or more different strains of FMDV. 8.The DNA vaccine of claim 1, wherein the modified FMDV 3C proteasefurther comprises a C142 substitution.
 9. The DNA vaccine of claim 1,formulated for injection, oral administration, intranasaladministration, topical administration, electroporation, gene gunadministration, transfection, or liposome-mediated delivery.
 10. The DNAvaccine of claim 1, wherein the vaccine is a marker vaccine or a DIVAvaccine (Differentiating Infected from Vaccinated Animals).
 11. The DNAvaccine of claim 1, wherein the polynucleotide encoding a FMDV P1precursor polypeptide and the polynucleotide encoding a modified FMDV 3Cprotease are contained in at least one of a minicircle vector, areplication deficient adenovirus vector or a vaccinia virus vector. 12.The DNA vaccine of claim 1, wherein the modified FMDV 3C proteasecomprises a L127P substitution.
 13. The DNA vaccine of claim 12, whereinthe modified FMDV 3C protease further comprises a C142T substitution.14. A pharmaceutical composition, comprising: a polynucleotide encodinga FMDV P1 precursor polypeptide, a polynucleotide encoding a modifiedFMDV 3C protease, wherein the modified FMDV 3C protease comprises a L127amino acid substitution of a wild-type FMDV 3C protease; and apharmaceutically acceptable carrier.
 15. A method for inducing an immuneresponse against foot-and-mouth disease virus (FMDV) in a subject,vaccinating a subject against FMDV, or reducing severity of an FMDVinfection in a subject, comprising administering to said subject aneffective amount of a composition comprising: a polynucleotide encodinga FMDV P1 precursor polypeptide; and a polynucleotide encoding amodified FMDV 3C protease, wherein the modified FMDV 3C proteasecomprises a L127 amino acid substitution of a wild-type FMDV 3Cprotease, wherein the FMDV P1 precursor polypeptide is expressed andproteolytically processed by the co-expressed modified FMDV 3C protease.16. The method of claim 15, wherein the modified FMDV 3C proteasefurther comprises a C142T substitution.
 17. The method of claim 15,wherein the composition is administered by subcutaneous injection,intradermal injection, intramuscular injection, jet injection, orally,intranasally, topically, by electroporation, gene gun, transfection, orliposome-mediated delivery.
 18. The method of claim 15, wherein thecomposition is administered prophylactically to prevent or amelioratethe effects of a future infection, therapeutically to treat or empowerthe immune system of an infected subject, or both.
 19. The method ofclaim 15, wherein the composition is administered to the subject in asingle dose, a two dose schedule, or a multiple dose schedule.
 20. Themethod of claim 15, wherein the FMDV is one or more of FMDV 0 serotype,A serotype, C serotype, Asia 1 serotype, SAT1 serotype, SAT2 serotype orSAT3 serotype.
 21. The method of claim 15, wherein the subject is a cow,pig, sheep, goat, water buffalo, yak, reindeer, deer, elk, llama,alpaca, bison, moose, camel, chamois, giraffe, hog, warthog, kudu,antelope, gazelle or wildebeest.
 22. The method of claim 15, wherein themodified FMDV 3C protease comprises a L127P substitution.
 23. The methodof claim 22, wherein the modified FMDV 3C protease further comprises aC142T substitution.